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1. IntroductionAshwagandha (Withania Somnifera, WS), belonging to family Solanaceae, is an Ayurvedic herb also known as Indian winter cherry and Indian ginseng that has been traditionally known since ancient times in India for its numerous beneficial health activities. WS is one of the most important herbs in Ayurveda, which has been used for >3000 years in stress management, energy elevation and improving cognitive health [1,2,3,4] and to lower inflammation, blood sugar levels, cortisol, anxiety, and depression [5,6]. The plant is an erect, grayish, evergreen shrub with long tuberous roots, short stems, ovate and petiolate leaves, and greenish axillary and bisexual flowers. The leaves, roots, stems and flowers bear medicinal values with 29 common metabolites derived from the leaves and root extracts [6,7]. To date, this medicinal plant has been found to have anti-epileptic, anti-inflammatory, anti-arthritic, anti-depressant, anti-coagulant, anti-oxidant, anti-diabetic, anti-pyretic efficacies along with palliative effects such as analgesic, rejuvenating, regenerating and growth-promoting effects [8].Despite its medical use from time immemorial in many parts of the world, the basic and mechanistic studies relating to the potential of WS extracts (WSE) has not been studied in the clinical realm until relatively recently. Thus, several additional Randomized Double-blind Placebo Control Trials have been formed for various clinical conditions (Table 1) ranging from weight management to schizophrenia. So far each of these studies showed significant effectiveness of WS used for intervention vs. control group. Most notably, together these studies revealed that in all of these studies WS was safe and tolerable (Table 1).In contrast to these illnesses, the role of WS in cancers was reported around 1992 [15]. WS was shown to impede the growth of new cancer cells, but not normal cells, help induce programmed death of cells by generating reactive oxygen species (ROS), and sensistize cancer cells to apoptosis [16,17,18]. Pre-clinical studies in several cancer types have shown up to 80% inhibition using combination chemotherapy [19]. Despite this progress, however, a comprehensive review of molecular mechanisms of the regulation by WS and its major component Withaferin A (WFA) is lacking. Herein, we provide a comprehensive review of the effects of WS vs. WFA on different cancers as well as their mechanistic role in decreasing the cancer growth and reducing toxicities resulting from radio and chemotherapies. 2. History of WSHerbal therapies have been extensively used in traditional medicine (including Ayurvedic and Chinese) since time immemorial. Medicinal plants contain different cytotoxic constituents that induce autophagy, necroptosis and apoptosis by influencing various proteins involved in the apoptotic pathway [20]. Although the various beneficial effects of the WS plant and its root, stem and leave extracts are known historically, the first published literature on the antibacterial principle of WS dates back to 1958 by Kurup PA [21]. Later, Malhotra et al. reported the effects of the total plant extract on central nervous system, smooth muscles, cardiovascular system, respiration and skeletal muscles in the 1960s [22,23].The chemotherapeutic properties of the substances isolated from the leaves was found in the literature but it was Dhalla et al. who first reported the chemical studies of the leaves of WS [24,25]. In 1973, the root extract of WS was isolated as a C28 steroid lactone as 5, 20α-Dihydroxy-6α, 7α-epoxy-1-oxo-with a-2, 24-dienolid whose structure was found to be similar to a withanolide isolated from the roots of Withania coagulants [26].For the first time, Chakraborti et al. reported the variations in the antitumor constituents of the WS dunal and the in vivo growth inhibitory effects of the root extracts of plant in a transplantable mouse tumor, Sarcoma 180 in 1992 [15,27]. Thus, intraperitoneal injection of the root alcoholic extract 400 to 1000 mg/kg body weight in BALB/c mice daily post 24 h intradermal inoculation of 0.5 × 106 cells of S-180 resulted in tumor regression. Later the same group showed the radio-sensitizing and antitumor effects of the root extract in the sarcoma model [28]. It was not until 1996, that WFA’s radiosensitizer activity was reported that caused V79 cell survival reduction where 1-h pre-treatment at 2.1 µM dose before radiation significantly killed cells [18,29]. Later, the anti-carcinogenic activities of WFA was found to be effective in various types of cancer treatment both in vitro and in vivo. 3. WS Extracts and their Anticancer ActivityThe pharmacological activity of the commercially available herbal supplements of WS extracts is conferred by its various alkaloids as well as WFA. Since the European Food Safety Authority (EFSA) has classified WFA as toxic, its application in cancer therapy by killing tumor cells is immense. Based on the types of extracts of the different parts of the plant such as water extract, methanol/ethanol extracts mainly from leaves, stems and roots research has advanced in exploring the active constituents and their effects in cancer. Table 2 summarizes these plant parts and their efficacies in cancer therapy. Interestingly, the whole plant extract was found to increase cell proliferation, stem cell proliferation, WBC (white blood cells) content, in sharp contrast to using either root or stem extracts. The method of extraction i.e., using organic solvents such as methanol or aqueous extracts for either roots, stems or leaves did not change the anti-cancer mechanism of the extracts. Radio-sensitization to altered expression of inflammatory cytokine genes, enhancing generation of reactive oxygen species, inhibiting NF-κB activation. Notably, leaf extracts showed alteration of genes involved in cell cycle. 4. Active Components in WS ExtractThe major biological compounds, which are found from different parts of the plant, are C-28 steroidal lactone triterpenoids, also known as withanolides (approx. 40 unique compounds), which are mostly comprised of withanolide A, WFA, withanone and withanolide D. The structure of withanolides is based on an ergostane backbone comprised of a lactone ring at the C-8 or C-9 side chain. Apart from withanolides, alkaloids, flavonoids, steroids, withanamides, withanosides, withanolide glycosides with a glucose at carbon 27 also known as glycol-withanolides (sitoindoside IX and X), steroidal saponins containing an additional acyl group (sitoindoside VII and VIII), cuscohygrine, anahygrine, salts, coagulins and other nitrogen containing compounds are also found in the various plant parts. Alkaloids, for example isopelletierine, cuscohygrine, anahygrine, tropine, and withanine, are relevant phytochemicals of WS. Apart from the broad-spectrum therapeutic activity, the extracts of the leaves, roots, stems and fruits as well as the isolated withanolides, Withaferin, have emerged as a potent anti-carcinogenic agent in lung, breast, colon, cervical, brain, prostate and other cancers. Particularly, WFA, Withanolide D, Withalongolide A and its triacetate derivatives have been found to possess anti-carcinogenic activities (Figure 1) [54,55]. WFA acts as an inhibitor of the chaperon p97 and it along with its analogues can be a proteostasis modulator by retaining p97 activity and cytostatic activity in vitro [54]. Recently, Motiwala and co-authors have reported the synthesis and cytotoxicity of semisynthetic Withalongolide analogues where 24 compounds were tested on five cell lines (JMAR, MDA-MB-231, SKMEL-28, DRO81-1, and MRC-5) [55]. The other constituents including WFA have hepatoprotective, cardio-protective, immunosuppressive, anti-inflammatory, neuroprotective, anti-oxidative and anti-microbial activities. WFA treatment leads to apoptosis, evasion of anti-growth signaling and immune system along with sustained proliferative signaling and interactions with the tumor microenvironment [56]. The recent updates on the anti-carcinogenic effects of WFA on various cancers (breast, colon, prostate, lung, ovarian along with renal, head and neck, pancreatitis, liver and skin cancers) are summarized in Table 3 along with their mechanisms of action and plausible pathways. 5. Role of WS and WFA in Various Cancers 5.1. Breast CancerLuminal A/B (estrogen-receptor and/or progesterone-receptor positive and HER2 (human epidermal growth factor receptor 2) negative or positive) and triple negative/basal like (TNBC) (estrogen-receptor, progesterone-receptor and HER2 negative) are the molecular subtypes of breast cancers where the effects of WFA have been extensively studied [64,65]. When studied for the proapoptotic response of WFA, it was found the phytochemical downregulated the estrogen receptor-α (ER-α) protein in MCF-7 cells. This effect reversed in presence of 17β–estradiol (E2). Thus WFA acts as anti-estrogen and p53 knockdown partially reduce WFA-mediated proapoptotic effects [66]. Moreover, in therapy-resistant TNBC, WFA studied for invasive and metastatic effects showed not only anti-metastatic behavior in nM concentrations but also lower extracellular matrix (ECM) gene expression and transcriptional patterns towards non-invasiveness by targeting uPA (urokinase-type plasminogen activator) signaling cascade [67]. In cancer patients using cancer genome atlas, WFA was found to suppress TNBC gene expression compared to the luminal cancers [68]. These studies provide invaluable evidence of the anti-cancer effects of WFA in both luminal and TNBC via diverse pathways.Although WS leaf, root and fruit extracts have shown curative effects in multiple diseases, the exact mechanism behind the action of these extracts is not well understood. Mohan et al. [69] were the first to report that WFA can bind to vimentin intermediate filaments causing them to aggregate in the cytoplasm leading to apoptosis in the MCF-7 cell line. Yang et al. later reported that treatment with the root extract leads to inhibition of mammary cancer metastasis and epithelial to mesenchymal transition via vimentin inhibition [70].Widodo et al. reported that WFA selectively activated p53 in tumor cells treated with the leaf extract of Ashwagandha [71] leading to growth arrest and apoptosis. Amongst other mechanisms, apoptosis due to generation of reactive oxygen species (ROS) by WFA has been widely reported. Hahm et al. demonstrated both in vitro and in vivo that the role of WFA in inducing apoptosis is mediated by ROS generation due to the inhibition of mitochondrial respiration. MDA-MB-231 and MCF-7 cell lines showed increased ROS production upon treatment as opposed to the normal human mammary epithelial cells (HMEC) [72] which did not increase ROS production. The molecular phenomenon behind the WFA-induced ROS-mediated apoptosis was due to the inhibition of oxidative phosphorylation and complex III activity accompanied with apoptotic histone-associated DNA fragment release in the cytosol as evidenced by significant reduction in the ectopic expression of Cu, Zn-superoxide dismutase in the aforementioned breast cancer cell lines. This mechanism was tested in mitochondrial DNA-deficient Rho-0 variants of MDA-MB-231 and MCF-7 cells where no apoptosis or related mitochondrial stress were observed [72]. In another study, Ghosh et al. demonstrated that not only apoptosis, but also paraptosis (non-apoptotic programmed cell death) is caused by WFA-induced production of ROS. These observations were supported by the formation of large cytoplasmic vacuolar structures due to the fusion of mitochondria and endoplasmic reticulum dilation in human breast cancer cell lines (MDA-MB-231 and MCF-7) along with downregulation of the endogenous paraptosis inhibitor, Actin Interacting Protein-1 (Alix/AIP-1), upon WFA treatment [73].Widodo et al. have used hammerhead ribozymes (catalytic RNAs) to identify genes and targets involved in WFA-mediated cellular cytotoxicity. MCF-7 breast cancer cells were infected with a retroviral vector carrying a randomized ribozyme library. The cells were then treated with WFA and ribozymes were retrieved from the surviving cells. Targets identified were validated using shRNA knock down of the target genes as well as bioinformatics pathway investigation. shRNA studies have shown 4 genes (TPX2 (Targeting protein for Xklp2), ING1 (inhibitor of growth protein 1), TFAP2A (transcription factor AP-2 alpha) and LHX3 (LIM/homeobox protein) are involved in WFA and withanone induced cellular cytotoxicity. Silencing these four genes led to decreased killing of cancer cells by 20–40% by the extract. Using bioinformatics and systems biology approach, the group identified p53 and apoptosis pathways to be involved in WFA-mediated cytotoxicity (Figure 2). Network interaction analysis showed 4 gene clusters: CDK4 (cyclin-dependent kinase 4), TFAP2A, CDKN1A-p21 (cyclin dependent kinase inhibitor 1A) and ING1 linked by p53 and PCNA (proliferating cell nuclear antigen). They hypothesized that the extract-mediated cellular cytotoxicity through mitochondrial stress and DNA damage pathway leads to activation of ROS-mediated cellular signaling. The group found an increase in γ-H2AX and number of cells expressing the phosphorylated form which is a marker for DNA damage in WFA treated MCF-7 cells. In addition, an increased tolerance to WFA treatment on p21-/- cells confirmed the role of CDKN1A-p21 in WFA-mediated cytotoxicity. ROS was detected in MCF-7 cells treated with the extract, withanone or withaferin. As ROS is well known to affect mithochondrial membrane potential, they found a change in mitochondrial membrane potential and altered mitochondrial morphology in WFA treated cells. Therefore, the study concludes that Ashwagandha extract and Withanone mediate selective killing of cancer cells by induction of ROS production and mitochondrial damage and hence, can be used for effective and safe cancer therapy [74]. Recently, it has been reported that mitochondrial dynamics are involved in breast cancer apoptosis when treated with WFA. [75]. Additionally, although the levels of XIAP (X-linked inhibitor of apoptosis protein), cIAP-2 (cellular inhibitor of apoptosis protein-2) and Survivin proteins were found to be reduced in MDA-MB-231 and MCF-7 cells when treated with WFA, in a MDA-MB-231 xenograft model, WFA-mediated inhibition was associated only with Survivin protein suppression thus highlighting the importance of Survivin suppression in WFA-induced apoptosis. These results provided a novel insight into the molecular mechanism of WFA-induced apoptosis in human breast cancer cells [76]. 5.2. Colorectal CancerColorectal cancer (CRC) is divided into inherited, familial and sporadic types, which represents 70% of all CRC cases. Histologically, CRC is classified into adenocarcinoma (representing 95% of all cases), lymphoma and squamous cell carcinoma. CRC usually develops from pre-neoplastic lesion due to 2 major genetic alterations either chromosomal instability or microsatellite instability. The major molecular mutations commonly found in CRC include, p53 mutations (50%), KRAS (K-ras) mutations (25–60%), BRAF (B-Raf) (10%) and PIK3CA (phosphatidylinositol 3-kinase catalytic subunit alpha) (10–30%) [77].Whereas the role of WFA has been examined in colorectal cancer cells, the investigations using WS are limited despite the latter’s use as a dietary supplement, i.e., it is taken orally. Major component of WS was found to have an anti-carcinogenic effect on colorectal tumors through alterations of multiple signaling pathways. The anti-cancer effects of WFA on the proliferation and migration of colorectal cancer cell lines have been cited due to reduced transcriptional activity of STAT3 (signal transducer and activator of transcription 3). Also, in HCT116 xenograft tumors in a Balb/c nude mouse model, the authors found a regression in the growth of the tumors thus proving the potential of WFA as a STAT3 inhibitor [78]. Further, Notch-1 signaling pathway plays a crucial role in the development of colon cancer and WFA has been shown to inhibit this signaling including Akt/NF-κB/Bcl-2 pro-survival pathways. A molecular link between Notch/Akt/mTOR signaling was established and WFA inhibition of Notch-mediated signaling aided in JNK-( c-Jun N-terminal kinase) mediated apoptosis in colon cancer cell lines, HCT-116, SW-480 and SW-620 [79,80].The chemopreventive effects of WFA have been studied by Chandrasekaran and colleagues in spontaneous and inflammation-associated colon cancer transgenic adenomatous polyposis coli (APCMin/+) and azoxymethane/dextran sodium sulfate (AOM/DSS) induced mice models respectively. WFA was orally administered at doses of 3 and 4 mg/kg and the authors found 59% reduction of tumor and polyp initiation and progression in the WFA treated mice compared to the controls [80]. WFA downregulated expression of inflammatory markers in these tumors such as IL-6, TNF-α, COX-2 along with pro-survival markers such as pAkt, Notch1 and NF-κβ [80]. These results are in agreement with the priming effect of the root extract of the herb in chemotherapy that modulated mitochondrial function, thus proving the priming effect of the root extract as a potential mechanism through increased ROS [33]. 5.3. Prostate CancerWFA has been utilized as a therapeutic agent for prostate cancer therapy where it acts as a regulator of G2/M phase transition of the cell cycle through the upregulation of phosphorylated Wee-1, phosphorylated histone H3, p21, Aurora B and the downregulation of A2, B1 and E2 cyclins and phosphorylated Cdc2 (Tyr15) [81]. Among various mechanisms involved in prostate cancer initiation and progression, activated protein kinase B/Akt plays a key role where the inactivation of the tumor suppressor PTEN gene (phosphatase and tensin homologue) leads to activation of Akt and subsequent development of prostate tumors [82] (Figure 3). Moselhy et al. reported the chemopreventive action of WFA in the Pten conditional knockout mouse (Pten-KO) model with constitutively activated Akt signaling. Oral administration of WFA with a dose of 3–5 mg/kg inhibited the activation of Akt and facilitated the FOXO3a-(Forkhead box O3a) mediated activation of Par-4 (prostate apoptosis response-4) leading to delayed tumor progression in preclinical prostate cancer models. Thus, WFA has been effective in up-regulating Par-4 and FOXO3a proteins (PI3K/Akt pathway regulated) in Pten-KO mice with a promising outcome for patients with Akt-activating mutations [83,84].In prostate cancer cells, switching from autophagy to apoptosis has been found after treatment with a semi-synthetic analogue, 3-azido derivative of WFA (3-AWFA) due to the pro-apoptotic protein PAWR-mediated suppression of BCL2 [85]. Androgen-independent prostate cancer cell lines (PC-3 and DU 145) were tested where 3-AWFA treatment lead to conversion of cytosolic MAP1LC3B-I/LC3B-I to MAP1LC3B-I/LC3B-II (microtubule-associated protein-1 light chain 3β) and reduction of the autophagy substrate, SQSTM1 (sequestosome 1) [85]. The 3-AWFA molecule has also been reported separately by Rah et al. as a novel matrix metalloproteinase-2 (MMP-2) inhibitor. The authors investigated the mechanistic role of 3-AWFA as an extracellular Par-4 modulator on the invasion and angiogenesis of PC-3 and DU-145 cells compared to non-prostate cancer cells (HeLa and A549) [86]. Androgen receptor (AR) function suppression or blocking androgen signaling is an important therapy for androgen-dependent or independent therapy. Srinivasan et al. reported that treatment with WFA leads to apoptosis by a Par-4-dependent mechanism through caspase signaling and inhibition of NF-κB activity [57]. Nishikawa et al. reported that a treatment with 2 µM WFA resulted in cell death in androgen-independent prostate cancer cells (PC-3 and DU-145) compared to the androgen-sensitive cells (LNCaP) and to non-prostate normal fibroblasts (TIG-1 and KD). Compared to TIG-1 and LNCaP, the mRNA levels of c-Fos and 11 HSPs (heat-shock proteins) were increased in the WFA treated PC-3 and DU-145 cells but the expression of anti-apoptotic proteins c-FLIP (L) was found to be reduced [87]. 5.4. Lung CancerLung cancer is broadly classified into non-small cell lung cancer (NSCLC) representing 85% of all lung cancer cases, and small cell lung cancer (SCLC) (15%). Lung cancer is also classified based on the driver oncogenic mutation such as EGFR (epidermal growth factor receptor) mutation (20%), ALK (anaplastic lymphoma kinase) rearrangement ( T) in vivo lung tumors in NOD/SCID (Non-obese Diabetic/severe combined immune deficiency; NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJl) mice [89]. Others have shown that WFA is effective for targeting KRAS mutant NSCLC cell lines in vitro e.g., A549 (c.34G > A), H3528 (c.34G > T) and H460 (c.183A > T) [90].In case of lung cancer treatment, WFA was reported to induce apoptosis in the NSCLC cell line A549 using annexin V/PI assay [91]. In addition, WFA inhibited the proliferation of A549 cells, as the number of cells in the G0/G1 phase was higher in treated cells. WFA caused a dose-dependent decrease in pAkt/Akt, the anti-apoptotic protein Bcl-2, and increases in Bax and cleaved caspase-3. Therefore, WFA was shown to have an anti-proliferative and pro-apoptotic action on A459 cells via suppression of PI3/Akt pathway [91] (Figure 4). Liu et al. have shown that WFA is selectively cytotoxic to A549 lung cancer cells at IC50 of 10 µM, whereas it is non-toxic to control normal lung cells WI-38 and PBMC. WFA increased cell apoptosis in annexin V/PI assay and Bax/Bcl ratio. Using JC-1 stain, WFA was found to induce a change in mitochondrial membrane potential indicating mitochondrial damage. WFA treated cells showed higher levels of caspase 3 and caspase 9 indicating the activation of mitochondrial pathway of apoptosis. WFA treatment showed a time-dependent increase in ROS production beginning 6 h after treatment and increasing until it reached a 5-fold increase after 24 h. Adding the anti-oxidant N-acetyl cysteine (NAC) to WFA treated A549 cells abrogated ROS production, apoptosis, and enhanced cell viability compared to groups treated with WFA alone, confirming that ROS production is an essential mechanism in WFA-mediated cytotoxicity [92].Kyakulaga et al. have shown that WFA inhibits invasion and metastasis of NSCLC by inhibiting epithelial to mesenchymal transition (EMT). WFA was found to be cytotoxic in NSCLC cell lines with the metastatic cell line H1299 being more sensitive than the non-metastatic cell line A549. When the cells were treated with a sub-toxic dose of WFA, cell adhesion was reduced to 60–70% of the untreated cells. Moreover, WFA significantly reduced wound healing, migration and invasion of H1299 and A549 cells. WFA treatment abolished the in vitro induction of EMT using TGF-β (transforming growth factor β) and TNF-α. WFA treated cells showed no increase in EMT markers including: vimentin, claudin, fibronectin and snail. In addition, there was no change in cell morphology, loss of E-cadherin or increased vimentin expression. Mechanistically, WFA reduced the levels of smad-3 phosphorylation and nuclear localization of smad-2/3 which are known to mediate TGF-β induced EMT. In addition, WFA inhibited the degradation of Iκ-Bα phosphorylation and nuclear translocation of NF-κb leading to the inhibition of TNF-α-mediated EMT [93].Kunimasa et al. showed that WFA in combination with glucose metabolism targeted therapy could be used as an effective treatment for tyrosine kinase inhibitor (TKI) drug-tolerant cancer cells. EGFR mutant lung cancer cell lines treated with gefitinib developed drug tolerance persisters (DTPs) characterized by increased senescence (CD133 low) and stemness (marked by CD133 high population). Senescent cells show a phenotype called SASP (senescence-associated secretory phenotype) and can communicate with other cells through secreted factors. Conditioned media from gefitinib treated SASP increased the number of CD133high cancer stem cells (CSCs). Gefitinib-tolerant DTPs were resistant to conventional cancer therapies such as cisplatin and pemetrexed. The group proposed using glucose metabolism targeting therapy (as senescent CD133 low cells are characterized by increased glucose metabolism) in combination with WFA for targeting CSCs. Glucose metabolism targeting therapies (e.g., phloretin) and WFA were found to possess an anti-tumor activity against gefitinib DTPs. Further, an in vivo gefitinib-induced DTP model was generated by treating a xenograft tumor with gefitinib until relapse occurred and the tumor continued to grow in the presence of the drug. Then, WFA and glucose transport inhibitor (phloretin) treatment was introduced causing a dramatic reduction in tumor size, suggesting that the combination of WFA and metabolism targeting therapies could be an effective therapeutic strategy against EGFR resistant lung cancer [94]. 5.5. Ovarian CancerAmongst other cancers, WFA has also shown tremendous efficacy in ovarian cancer treatment particularly in combination therapy. Combination of WFA and cisplatin proved to be an effective treatment of refractory ovarian cancer (OC) by reducing the number of Aldehyde Dehydrogenase ALDH+ CSCs. Immunostaining showed that ALDH expression in the ovarian cortex was higher than its expression in ovarian surface epithelium (OSE) in borderline (BL) and high-grade (HG) ovarian tumors indicating the role of ALDH1 in invasion and metastasis of OC. In sphere formation assay, ALDH+ CSCs were isolated from the OC cell line A2780 spheroid formation was measured and found to be significantly reduced by WFA treatment at a dose of 1.5 µM. Cisplatin (CIS) treatment reduced the spheroid formation, albeit non-significantly. Cisplatin and WFA combination significantly reduced spheroid formation when compared to control, cisplatin only or WFA only treatment. In an orthotopic OC mouse model, WFA treatment significantly reduced ALDH+ CSC population, whereas Cisplatin treatment increased CSC population. The combination of CIS and WFA showed the highest reduction in ALDH+ CSCs (as shown by immunostaining and western blotting for ALDH1). WFA also reduced the levels of securin, an oncogene that is associated with cancer stemness. Cisplatin increased the levels of securin, indicating the enrichment of CSC population and this effect was reversed when the combination of CIS and WFA was used [19]. Kakar et al. have shown the WFA and DOXIL combination can be used for the elimination of ALDH+ CSCs responsible for relapse of OC patients [95]. 5.6. Other CancersApart from the major cancers described above, WFA has been reported to show potent anti-cancer properties in several other cancer types, such as gastric cancer, papillary and anaplastic thyroid cancers, cervical cancers, melanomas, renal carcinoma and promyelocytic leukemia.In gastric cancer, WFA inhibited proliferation of human gastric adenocarcinoma (AGS) by inducing G2/M cell cycle arrest and apoptosis [96]. In addition to killing normal cancer cells, WFA was shown to target cancer stem cells and metastatic cancer cells. It was reported that in lymph node metastatic gastrointestinal cell line (UP-LN1), WFA reduced the CD44high/CD24low floating (F) cell proliferation with greater apoptosis via downregulation of CXCR4/CXCL12 and STAT3/interleukin-6. The targeting ability of WFA on CSCs and mCSCs has been validated in NOD/SCID mouse xeno-transplantation [97].In papillary and anaplastic thyroid cancers, combination of the multikinase-targeted inhibitor Sorafenib with WFA has been found to act synergistically via multiple mechanisms. PARP (Poly (ADP-ribose) polymerase cleavage, caspase-3 cleavage, BRAF/Raf-1 downregulation and inhibition of heat shock protein resulted from the combination therapy in vitro (B-CPAP, SW1736, human papillary and anaplastic thyroid cancer cell lines) [98].In cervical cancer treatment, Munagala et al. showed for the first time that WFA restores the inactivation of the tumor suppressor p53 protein thus downregulating human papilloma virus (HPV) expressing E6/E7 oncogenes both in vitro and in vivo was found to be 0.45 ± 0.05 µM with altered expression levels of Bcl2, Bax, caspase-3, cleaved PARP. In addition, WFA lowered the levels of STAT3 and its phosphorylation at 705Tyr and 727Ser [99].In case of melanoma, Mayola et al. tested WFA in four human melanoma cell lines and found WFA induced apoptotic cell death with IC50 ranging from 1.8 to 6.1 µM with the involvement of mitochondrial pathway. WFAs downregulated Bcl-2, with Bax mitochondrial translocation, cytochrome c release into the cytosol, activation of caspase 3 and 9 and fragmentation of DNA [100].Endoplasmic reticulum stress was found to be the driving force in human renal carcinoma cells when treated with WFA. Dose-dependent WFA-induced apoptotic cell death in renal carcinoma kidney cell lines and induction of ER (endoplasmic reticulum) stress markers such as phosphorylation of eIF-2α (eukaryotic initiation factor-2α), XBP1 (X-box binding protein 1) splicing, upregulation of glucose-regulated protein (GRP)-78 and CHOP (CAAT/enhancer-binding protein-homologous protein). Mechanistically it was demonstrated by the pre-treatment of NAC (N-acetyl cysteine), the inhibition of WFA-mediated ER stress protein by ROS generated cell death [101].Recently, Yu and co-authors investigated the action and mechanism of WFA on cancer cells with and without telomerase. Maintenance of telomere length by activation of telomerase or ALT (Alternative mechanism of Lengthening of Telomeres) led to overcoming replicative mortality by cancer cells and WFA was found to have stronger cytotoxic effects on ALT cells by telomere dysfunction, DNA damage, upregulation and inhibition of ALT-associated promyelocytic leukemia nuclear bodies in these cells. It was also found from computational and experimental analyses for effect on ALT mechanism, WFA led to Myc-Mad-mediated transcriptional suppression of an MRN complex protein (NBS-1) [102]. The overall effects of WS on different cancers are shown in Figure 4. 6. WS and Cancer Chemotherapy–Induced Toxicities.The traditional chemotherapies induce many adverse effects including those affecting functions of several organs such as heart, liver, kidney, etc. In myocardial ischemia reperfusion (MI/R) injury WFA was found to increase cellular survival in simulated injury and in H2O2-induced cell apoptosis along with inhibition of oxidative stress. Thus, via upregulation of SOD2, SOD3, Prdx-1 by H2O2, WFA treatment leads to inhibition of the antioxidants and Akt-dependent improvement of cardiomyocyte caspase-3 [103]. Also, pre-treatment with WFA (10 mg/kg) induced a protective role as manifested by the lowering CYP450-mediated reactive metabolites resulting in oxidative stress in Bromobenzene-mediated liver and kidney damage. Oxidative stress and cytokines were reduced in addition to the prevention of mitochondrial dysfunction and restoring the balance between Bax/Bcl-2 in the WFA pre-treatment mice group [104]. Further, WFA reduced acetaminophen-induced liver toxicity in mice in Nrf2-dependent manner, which is a stress-responsive transcription factor and a validated chemoprevention target. In this study, Nrf2 signaling was induced by WFA in a non-canonical Keap-independent, Pten/PI3k/Akt-dependent manner [105]. Moreover, WFA decreased Cerulein-induced acute pancreatitis caused by oxidative stress and inflammation [106]. Lastly, WFA was found to induce antifibrotic activity in scleroderma by suppressing pro-inflammatory fibrosis involving TGF-β/Smad signaling and conversion to myofibroblasts, a FOXO3a-Akt-dependent NF-κβ/IKK-mediated inflammatory cascade [107]. A recent study involved the application of tumor targeting silver nanoparticles (Ag NP), which induce NP-related toxicity in macrophages. However, when NPs were administered along with the root extract of WS (35 mg/kg), the latter induced a significant reduction in toxic effects in rats [108].Despite these basic and mechanistic studies, the potential of WS extracts as diet supplement has not been studied in the clinical realm except the single report that involved an open label prospective non-randomized trial on 100 breast cancer patients receiving chemotherapies that showed the potential of WS (used as complementary)-mediated decrease of treatment related fatigue and improved quality of life [109]. 7. Concluding RemarksThe 2014–2023 World Health Organization (WHO) strategy aims to alleviate healthcare issues by providing traditional medicines as part of their affordable and effective alternative medicines to culturally diverse populations [110]. For a global implementation of these alternative herbal medicines, detailed and thorough evidence-based approaches should be executed to study their safety, efficacy and quality [111]. As reviewed herein, the recent clinical trials using randomized double-blind placebo control designs using WS extracts have shown that at specified dosage ranging from 200 mg/kg to 1000 mg/kg WS was not only effective, but most importantly at these dosages WS was safe and well tolerated. Further, numerous studies have shown anti-cancer efficacy using either WS or its major component WFA in human cancer cells lines and in murine models. The safety of WS in humans and the potential therapeutic efficacy seen in pre-clinical studies with the underlying diverse molecular pathways suggests the potential of WS and WFA use in patients with different cancers. There exist at least two different ways WS can be utilized against neoplastic diseases. First, given the safety record of WS, it can be used as an adjunct therapy that can aid in reducing the adverse effects associated with radio and chemotherapy due to its anti-inflammatory properties. Second, WS can also be combined with other conventional therapies such as chemotherapies to synergize and potentiate the effects due to radiotherapy and chemotherapy due to its ability to aid in radio- and chemosensitization, respectively. Taken together, all evidence to date indicate the potential of WS or WFA in cancer management. However, this needs to be validated in clinical studies prior to translation into the clinical realm.

Abstract

Microorganisms. 2023 Mar; 11(3): 713. PMCID: PMC10051562,1 ,1 ,2 ,3,4 and 1,5,*Josep M. Llibre, Academic EditorAbstract

In recent years, LIPUS has been found to have a wide range of biological effects on tissues and have been applicated in many ways in the field of therapeutic medicine, such as promoting bone-fracture healing (2), accelerating soft-tissue regeneration (4), and inhibiting inflammatory responses (5) and so on. Thus, here reviews the current applications of LIPUS in medical activities of rehabilitation medicine. Still, there are many challenges for this relatively new application, and the achievements using it promises to go far beyond the present possibilities.Clinical therapeutic proceduresWhen treatments were performed, the target tissues should be placed in a suitable position with local anesthesia or without anesthesia, and the ultrasound probe head is usually suspended from an articulating arm for flexible movement. Applied locations, treatment duration time, and treatment cycles are chosen according to the kind of equipments and disease applications.For therapeutic purposes, it is vital to pass the energy into the human tissues completely. So, a number of methods are used to couple the sound into the tissue. Aqueous gel may be used between the source’s head and the tissue skin when the tissue surface is relatively flat and the probe head is plane. Otherwise, water may provide a better coupling medium for awkward tissue geometries. Besides, it is important that the couplant is degassed to prevent the occurrence of cavitation.Clinical applications of LIPUSCurrently, LIPUS is accepted to promote bone-fracture healing (2), accelerating soft-tissue regeneration (4), and inhibit inflammatory responses (5). Besides, it has made it as a tool to be used to enhance regeneration and tissue engineering, for example being used in oral and maxillofacial regions (31).Bone-fracture healing Corradi and Cozzolino first reported in 1952 that continuous wave ultrasound could stimulate the formation of bone callus in a radial fracture rabbit model, and it was proved by the same research group in the next year that the ultrasound wave was safe and could produce an increase in periosteal callus in eight patients, and this is the first evidence of application of ultrasound wave on fracture healing (32).In 1994 and 1997, Heckman and Kristiansen performed two rigorous, prospective, randomized, double-blind, placebo-controlled clinical trials and found that the rate of healing of fresh fractures is accelerated by non-invasive LIPUS (33,34). The first trial tested the efficacy of ultrasound on closed or grade-I open fractures of the tibial shaft, and the second trial tested the efficacy of ultrasound on dorsally angulated fractures (negative volar angulation) of the distal aspect of the radius. The patients in both trials had been imposed ultrasound stimulating device 30 mW/cm2 daily for 20 min at home for 10 weeks as an adjunct to conventional manipulation treatment with a cast. Results showed the specific ultrasound accelerate the healing of fractures and decrease the loss of reduction during fracture-healing, and there were no serious complications related to the use of the ultrasound device. These two clinical trials primarily promoted the U.S. Food and Drug Administration (FDA) approve the use of low-intensity ultrasound for the accelerated healing of fresh fractures in 1994 and for the treatment of established nonunions in 2000 (32).In a meta-analysis of six randomized controlled trials (RCTs) performed by Busse et al. in 2002 (35), LIPUS was found to have a significant effect on reducing the time to fracture healing for fractures treated nonoperatively, results showed that fracture healing time was significantly shorter in low-intensity ultrasound therapy groups than that in the control groups. In a review of the clinical evidence on LIPUS for fracture healing in 2008 (26), Pounder and Harrison found that typically widely used LIPUS (1.5 MHz ultrasound pulsed at 1 kHz, 20% duty cycle, 30 mW/cm2 intensity) could accelerate the healing time by up to 40% in fresh tibia, radius and scaphoid fractures, and that it was shown to be effective at resolving all types of nonunions of all ages. In a review searching for the evidence of LIPUS for in vitro, animal and human fracture healing in 2011 (36), Martinez de Albornoz et al. agreed that LIPUS can produce significant osteoinductive effects, accelerate the healing process and improve the bone-bending strength in vitro and animal studies. In a cohort study of 4,190 patients treated with LIPUS performed by Zura et al. in 2015, older patients (≥60 y) with fracture risk factors treated with LIPUS were found to exhibit similar heal rates to the population as a whole (37).But there was still a controversy about the LIPUS effects in fresh, stress fractures and in limb lengthening in human trials. In a systematic review and meta-analysis of seven human clinical trials on fresh fractures in 2012 (20), Bashardoust Tajali et al. found that the time of the third cortical bridging (increase in density or size of initial periosteal reaction) in radiographic healing was statistically earlier following LIPUS therapy in fresh fractures, but there was a paucity of sufficient studies of LIPUS’s beneficial effects on delayed unions and nonunions. In addition, LIPUS may not have a potential beneficial effects for the treatment of acute fractures in adults, and future trials should record functional outcomes and follow-up all trial participants in clinical practice (38).Soft-tissue regeneration The targets of LIPUS effects on soft-tissue regeneration cover a wide range of cells and organs, including fibroblasts (3), myoblasts (39), epithelial cells (4), chondrocytes and cartilage (40-45), inter-vertebral discs (IVDs) (46,47), ligaments (48-51), and tendons (52,53).In 2004, Zhou et al. examined the effects of daily application of LIPUS on the proliferation of primary human foreskin fibroblasts (3). In their study, fibroblast cell was observed to have an increase in the total cell number and an increase of bromodeoxyuridine incorporation after LIPUS (1.5 MHz ultrasound wave, 200 µs pulse modulated at 1 kHz, with an output intensity of 30 mW/cm2) stimulation, and LIPUS can induce stress fiber and focal adhesion formation. They concluded that LIPUS promotes cell proliferation via activation of integrin receptors and a Rho/ROCK/Src/ERK signaling pathway.Ikeda et al. investigated the effects of LIPUS on the differentiation of C2C12 cell, which is a subclone of C2 myoblasts originally isolated from the thigh muscle of C3H mouse (39). In their study, they found that mRNA expression of Runx2, Msx2, Dlx5, AJ18, and Sox9 was increased by the LIPUS stimulation (1.5 MHz at an intensity of 70 mW/cm2 for 20 min), whereas the expression of MyoD, C/EBP, and PPARγ was decreased. And LIPUS stimulation increased Runx2 protein expression and phosphorylation of ERK1/2 and p38 MAPK. They concluded that LIPUS stimulation converts the differentiation pathway of C2C12 cells into the osteoblast and/or chondroblast lineage via activated phosphorylation of ERK1/2 and p38 MAPK.Ikai et al. evaluated the effects of LIPUS on wound healing in periodontal tissues after mucoperiosteal flap surgery in beagle dogs (4). After the LIPUS treatment (a 200 µs burst sine wave of 1.5 MHz repeated at a frequency of 1.0 kHz, 30 mW/cm2, daily for 20 min, for a period of 4 weeks), the expression level of heat shock protein 70 (HSP70) was higher in the gingival epithelial cells of the LIPUS-treated tooth, and the regeneration processes of both cementum and mandibular bone were accelerated. They came with a conclusion that ultrasound could accelerate periodontal wound healing and bone repair.In 2002, Nishikori et al. found that LIPUS exposure (1.5 MHz with a 200 µs tone burst repeated at 1.0 kHz, 30 mW/cm2, 20 min per day) could promote synthesis of chondroitin sulfate, especially chondroitin 6-sulfate, although it did not significantly enhance cell number and stiffness (40). In vitro cell studies, LIPUS was demonstrated to have an effect on stimulating chondrocyte proliferation and matrix production (41-43). The potential mechanisms of LIPUS effects on chondrocytes may be associated with activation of MAPK/Erk pathway and the increase of the anabolic factor (TIMP-1)/catabolic factor (MMP-3) ratio (44,45).LIPUS may also have effects on treating intervertebral disc herniation and delaying the progression of disc degeneration. In 2008, Omi et al. found that LIPUS stimulation could significantly activate TIMP-1 and monocyte chemoattractant protein-1 (MCP-1) in nucleus pulposus cells and macrophages at both the protein and gene levels (46). And in 2009, Kobayashi et al. found that LIPUS could upregulate the cell proliferation and proteoglycan sythesis in human nucleus pulposus cells via enhancement of several matrix-related genes (47).Takakura et al. found that LIPUS (30 mW/cm2, 20 min daily) is effective for enhancing the early healing of medial collateral ligament injuries in rats in 2002 (48). And Warden et al. found that LIPUS could accelerate ligament healing in a controlled laboratory study in adult rats (49). In a recently published paper, Hu et al. found that LIPUS can facilitate osteogenic differentiation in human periodontal ligament cells, the underlying mechanism may be associated with upregulation of Runx2 and integrin beta1 (50), and so involved p38 MAPK pathway signaling (51).In addition, bone-tendon healing can also be accelerated under the LIPUS treatment, both in the partial patellectomy model in rabbits (52) and the transosseous-equivalent sheep rotator cuff model (53).Inhibiting inflammatory responses During injury or in the forming of the rheumatoid arthritis (RA) and osteoarthritis (OA), inflammatory plays an essential role in these progresses (54). Recent studies have demonstrated that LIPUS could inhibit inflammatory responses both in vitro and in vivo.In 2014, Nakao et al. reported that LIPUS could inhibit LPS-induced inflammatory responses of osteoblasts through TLR4-MyD88 dissociation (5). In their study, LPS induced mRNA expression of several chemokines including CCL2, CXCL1, and CXCL10 in both mouse osteoblast cell line (MC3T3-E1) and calvaria-derived osteoblasts (from newborn C57BL/6 mouse). After the LIPUS (1.5-MHz, 200 µs burst sine waves at 1.0 kHz, 30 mW/cm2) treatment, CXCL1 and CXCL10 mRNA induction were significantly inhibited, and LPS-induced phosphorylation of ERKs, p38 kinases, MEK1/2, MKK3/6, IKKs, TBK1, and Akt was decreased. LIPUS inhibited the transcriptional activation of NF-kB responsive element and interferon-sensitive response element (ISRE) by LPS, and LIPUS significantly inhibited TLR4-MyD88 complex formation in a transient transfection experiment. And Nakamura et al. investigated the effects of LIPUS on inhibiting inflammatory responses in vitro in the rabbit knee synovial membrane cell line (HIG-82), which was cultured in medium with or without IL-1β or TNF-α (55). The parameters of LIPUS they used in their study were: 15 min of single LIPUS exposure, 3 MHz with a spatial-average intensity of 30 mW/cm2 and pulsed 1:4 (2 ms on and 8 ms off). The proinflammatory cytokines significantly up-regulated cell proliferation, and LIPUS could significantly down-regulated this action.Nakamura et al. also investigated the effects of LIPUS on inhibiting inflammatory responses in vivo in the knee joints of animal models for RA using MRL/lpr mice (55). The LIPUS parameters were as same as that in the above mentioned in vitro study. In MRL/lpr mice, treated with LIPUS for 3 weeks, histological damage of knee joints and lesions were significantly reduced compared to the control, and COX-2-positive cells were markedly decreased in the knee joints treated with LIPUS compared to the control joints. In 2012, Engelmann et al. evaluated the effect of LIPUS and dimethylsulfoxide (DMSO) gel treatment on the expression of pro-inflammatory molecules in an animal model of traumatic muscle injury (56). Results showed that LIPUS associated with DMSO gel can attenuate TNFα, IL-1β, NF-kB protein levels and JNK phosphorylation in traumatic muscle injury.

IntroductionImmune checkpoint inhibitor (ICI) therapy has revolutionized the paradigm of cancer immunotherapy. Under normal physiological conditions, immune checkpoints are crucial to maintaining immune tolerance. However, in the tumor environment, tumor cells hijack these inhibitory mechanisms to avoid antitumor immune responses. ICIs are monoclonal antibodies that disrupt the engagement of immune checkpoints, which enables tumor-reactive T cells to overcome inhibitory mechanisms and mount effective antitumor immune responses [1]. The United States Food and Drug Administration (FDA) has approved ICIs that target cytotoxic T lymphocyte-associated protein-4 (CTLA-4), programmed cell death-1 (PD-1), and programmed cell death-ligand 1 (PD-L1) for the treatment of a wide variety of cancers [2]. Despite the clinical success of ICIs, advancing clinical applications of ICIs face challenges related to both efficacy and safety. Most cancer patients are unable to derive durable remission, while >50% of cancer patients develop immune adverse events after they receive ICIs [3]. The combination of multiple ICIs with other cancer therapies has improved cancer treatment by enhancing direct tumor killing and indirect antitumor immunity [4].The past two decades have witnessed exciting breakthroughs in the clinical translations of focused ultrasound (FUS) modalities for cancer treatment [5]. FUS concentrates extracorporeally generated ultrasound energy through the body to a tight focus with an exceptional spatial resolution (on the millimeter scale) and deep penetration depth. The focal point can be mechanically and electronically steered in three-dimensional space to form a sonication volume that conforms to the shape of the target. FUS therapy is often performed under the guidance of magnetic resonance imaging or ultrasound imaging [6]. As a promising therapeutic technology, FUS has the unique combined advantages of being noninvasive, nonionizing, nonpharmaceutical, spatially targeted, and deeply penetrating the body. Since 2017, five FUS modalities, including high-intensity focused ultrasound (HIFU) thermal ablation [7–11], HIFU hyperthermia [12], HIFU mechanical ablation [13–17], ultrasound-targeted microbubble destruction (UTMD) [18–20], and sonodynamic therapy (SDT) [21, 22], have been investigated in combination with ICIs for treating solid tumors in mouse models. The enhancement of antitumor immune responses by these FUS modalities demonstrated the great promise of FUS as a transformative cancer treatment modality to improve ICI therapy.In this review, we provide a brief introduction of ICI therapy basics and discuss the challenges facing ICI therapy. We then introduce each FUS-enhanced ICI therapy and summarize the therapeutic outcomes achieved by the combination therapy. Finally, we discuss the limitations of existing studies and provide future perspectives.Immune Checkpoint Inhibitor Therapy Basics and ChallengesICIs bind to immune checkpoints, including CTLA-4, PD-1, and PD-L1, and “release the brakes” on T cells, resulting in anticancer immune responses. CTLA-4 inhibits T-cell activation by attenuating T-cell receptor signaling through competing with the costimulatory molecule CD28 for binding to B7 ligands on antigen-presenting cells (APCs) [23]. PD-1 regulates T-cell activation through interaction with its ligand PD-L1. The engagement of PD-1 and PD-L1 results in a negative costimulatory signal and leads to T-cell apoptosis, anergy, and exhaustion [24]. Efficient ICI therapy requires reactivation and clonal expansion of antigen-experienced T cells present in the tumor microenvironment (TME) [25]. Initially, naive tumor-specific CD8 T cells are primed by antigen presentation by APCs (often referred to as immune priming) and activated in the presence of costimulatory pathways and cytokines. Tumor-specific CD8 T cells subsequently differentiate into effector T cells, undergo clonal expansion, traffic to the TME, and ultimately kill tumor cells. A subset of effector T cells can differentiate into memory T cells under the guidance of CD4 T cells and dendritic cells (DCs) to develop long-term immunologic memory against the tumor.The introduction of ICI therapy in the clinic has been considered to be a paramount achievement in cancer treatment in the last decade [26]. Since 2011, the FDA has approved ICIs targeting PD-1 (pembrolizumab, nivolumab, and cemiplimab), PD-L1 (atezolizumab, durvalumab, and avelumab), and CTLA-4 (ipilimumab). They have produced remarkable results regarding tumor control in many malignancies, such as melanoma, metastatic non-small-cell lung cancer (NSCLC), head and neck squamous cancers, urothelial carcinoma, gastric adenocarcinoma, mismatch-repair-deficient solid tumors, and classic Hodgkin lymphoma. Many clinical studies with ICIs are currently underway to test their efficacy in various other diseases.Despite the clinical success of ICIs, ICI therapy faces challenges related to both efficacy and safety. With regard to ICI efficacy, the majority of patients do not benefit from the treatment, and some responders relapse after a period of response. Ongoing studies indicate that both tumor cell-intrinsic and tumor cell-extrinsic factors contribute to the resistance mechanisms [27]. Tumor cell-intrinsic factors include lack of tumor-associated antigens (TAAs), ineffective antigen presentation, activation of oncogenic pathways, and insufficient interferon-γ (IFN-γ) signaling. Tumor cell-extrinsic factors are within the TME and include exhausted CD8 T cells, regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and other immunosuppressive cells and factors. With regard to safety, a significant number of patients on ICIs develop immune-related adverse events affecting almost every organ. Immune-related adverse events occur when ICIs result in an immune-based attack on normal tissue. These events, such as dermatitis, thyroiditis, pneumonitis, colitis, hepatitis, and nephritis, are unpredictable, heterogeneous, and in some instances life-threatening. Management of these adverse events remains a challenge [28]. These challenges call for concepts to maximize the clinical benefits of ICIs in combination with other therapies. An abundance of clinical trials are currently underway in evaluating the combination of ICIs with other immunotherapies, chemotherapy, radiotherapy, or targeted therapies. Strategies that can improve antigen presentation and immune recognition, reinforce the activity and infiltration of CD8 T cells, and reduce immunosuppression can potentially be combined with ICIs to improve the efficacy of ICI therapy [27]. Meanwhile, novel drug delivery strategies that enable the targeted delivery of ICIs within the TME have the potential to reduce the toxicities associated with ICIs [29, 30].FUS is a promising platform technology to be combined with ICIs to improve its efficacy and safety. Various FUS therapeutic modalities have been developed, and some of them have been used in the clinic for the treatment of various diseases (Table 1). Among them, HIFU thermal ablation has been approved by the FDA for the treatment of prostate cancer, uterine fibroids, bone metastasis, and essential tremor and has been used worldwide for the treatment of various diseases. Although other FUS modalities, including HIFU hyperthermia, HIFU mechanical ablation, UTMD, and SDT, have not been approved for clinical use, clinical studies are currently ongoing, with multiple studies already reported. Advances in the clinical applications of these FUS techniques have encouraged new studies to combine FUS with ICIs, as summarized in Table 2. The effectiveness of FUS-enhanced ICI therapy is often demonstrated by increased tumor infiltrated CD8 T cells, decreased tumor volume, and prolonged survival. The systemic immune response of ICI therapy can also be demonstrated by the presence of the abscopal effect, which occurs when the treatment not only shrinks the targeted tumor but also leads to shrinkage of untreated tumors elsewhere in the body. In the following, each FUS-enhanced ICI therapy is introduced.TABLE 1. Overview of different FUS modalities.TABLE 2. Summary of therapeutic ultrasound-enhanced ICI therapies.High-Intensity Focused Ultrasound Thermal Ablation-Enhanced Immune Checkpoint Inhibitor TherapyHIFU thermal ablation induces thermal coagulation by rapidly (in a few seconds) heating tissue at the focus to >60°C, often with high-intensity continuous ultrasound waves. Only tissue within the focal region is selectively ablated, while tissue in the ultrasound beam path is spared from ablation [31]. Compared with other local ablative therapies, such as ablative radiotherapy, radiofrequency ablation, and cryotherapy, HIFU thermal ablation is the only noninvasive and nonionizing ablation technique, allowing the procedure to be performed and repeated without the need for surgical implantation of applicators and concerns about radiation-induced toxicities. HIFU thermal ablation causes very few side effects to normal surrounding tissues, and patient comfort and safety are maximized [32]. HIFU thermal ablation has been widely applied for the treatment of a variety of solid tumors, as well as many other benign diseases in the clinic [32]. HIFU thermal ablation has been reported to increase the release of damage-associated molecular patterns (DAMPs) and TAAs, promote DC maturation, increase tumor-infiltrating lymphocytes, and change circulating immunosuppressive cytokine levels [33], suggesting the potential to improve ICI efficacy for tumors that do not respond well to ICIs.The first study on therapeutic ultrasound-enhanced ICI therapy was reported in 2017 by Silvestrini et al. [7]. They explored whether HIFU thermal ablation could be effectively incorporated with ICIs to boost antitumor immune responses in murine breast cancer models. Breast cancer is often resistant to most chemotherapies and molecular targeted therapies, including ICI therapies [7]. Half of the reported FUS-enhanced ICI studies summarized in Table 2 used murine breast cancer models. In Silvestrini’s study, systemic anti-PD-1 antibody (αPD-1) and local adjuvant, CpG, were administered prior to HIFU thermal ablation for immunotherapy priming. Only with initial immunotherapy priming, coincident HIFU thermal ablation and immunotherapy suppressed tumor growth in both treated and contralateral nontreated tumors and increased the survival rate [7]. The potential mechanisms for the enhanced antitumor response from this multistep protocol were proposed as follows [8]: immunotherapy priming expanded the number of tumor-infiltrating CD8+ T cells and macrophages. The subsequent HIFU thermal ablation released tumor antigens, inflammatory chemokines and cytokines, increased interferon stimulating genes, and altered the local macrophage phenotype. These effects led to cross-presentation and cross-priming mediated by macrophages and DCs, resulting in an effective abscopal response.The combination of HIFU thermal ablation with ICIs has also been explored to treat colorectal tumors since some of them are not suitable for ICIs [34]. Without immunotherapy priming, the combination of HIFU thermal ablation with ICIs and local adjuvants was able to produce therapeutic benefits in colorectal tumor-bearing mice [9]. HIFU thermal ablation was followed by direct injection of nanoadjuvants into the ablated site and intravenous injection of anti-CTLA-4 antibody (αCTLA-4). The nanoadjuvants were formed by loading poly (lactic-co-glycolic) acid nanoparticles with either a TLR7 agonist (imiquimod, R837) or TLR4 agonist (monophosphoryl lipid A, MPLA). This combination strategy increased the intratumoral CD8 T cell/Treg ratio, reduced MDSCs within tumors, achieved complete distant tumor eradication, prolonged mouse survival, and prevented tumor recurrence, indicating the generation of sustained immune memory against colorectal tumors. In contrast, none of the mice given HIFU thermal ablation plus either nanoadjuvants or αCLTA-4 survived [9], which suggests that additional agents (e.g., adjuvants, chemotherapeutics) may be required for the success of HIFU thermal ablation and ICI combination treatment. Another study reported that initiating αPD-1 treatment prior to versus shortly after HIFU thermal ablation with chemotherapy did not bear a marked difference in primary tumor growth in a murine breast cancer model [10].These studies suggest that the optimal protocol for HIFU thermal ablation-enhanced ICI therapy may depend on the tumor model, the type of adjuvants used, with or without immunotherapy priming, and the HIFU ablation protocol. HIFU thermal ablation as a combination therapy with ICIs has the potential limitation that excessive heat generation by HIFU thermal ablation may induce protein denaturation and inactivate antigen presentation [13]. Meanwhile, HIFU thermal ablation was also reported to increase tumor infiltrated MDSCs and Tregs at both directly treated and distant tumors, leading to inhibition of antitumor immunotherapy [11]. These negative effects highlight the complexity of combining HIFU thermal ablation with ICI therapy.High-Intensity Focused Ultrasound Hyperthermia-Enhanced Immune Checkpoint Inhibitor TherapyHIFU hyperthermia raises tissue temperature within the focal region to 40–45°C for up to 60 min. It is different from thermal ablation in that hyperthermia is not intended to produce substantial cell death directly. Instead, HIFU hyperthermia is often combined with chemotherapy and radiation therapy or used for local drug release in combination with temperature-sensitive nanoparticles [35]. HIFU hyperthermia can directly promote antigen cross-presentation and tumor-reactive T cell formation and expansion [36].Kheirolomoom et al. investigated the combination of HIFU hyperthermia with chemotherapy, CpG, and αPD-1 [12]. HIFU hyperthermia was utilized to control the release of temperature-sensitive liposomes loaded with a chemotherapy drug, doxorubicin (Dox). The liposome carrier was designed to minimize the severe cardiac toxicity of Dox and enhance its delivery efficiency to tumors. Dox released at HIFU hyperthermia-treated tumors enhanced the presentation of tumor-specific antigens at distant tumor sites. Similar to HIFU thermal ablation [7], only with immunotherapy priming by CpG and αPD-1, the combined HIFU hyperthermia, Dox-loaded liposomes, and αPD-1 treatment increased tumor infiltrated CD8 T cells and achieved complete tumor destruction in both treated and distant tumors as well as prolonged tumor-free survival. However, repeated Dox delivery by HIFU hyperthermia either with or without immunotherapy priming reduced the complete response rate, which was considered to be caused by rapid tumor cell death resulting from repeated Dox release that weakened the impact of local antigen and cytokine release. These findings highlighted the importance of the dosing of HIFU hyperthermia-mediated chemotherapy and the timing of immunotherapy to augment ICI efficacy for cancer treatment.These reported studies [7, 9, 10, 12] suggest that neither HIFU thermal ablation nor HIFU hyperthermia alone is sufficient to enhance ICI efficacy in murine tumor models. Both FUS modalities were found to enhance the release of TAAs and recruitment of CD8 T cells, but in the absence of additional stimuli (e.g., adjuvants, chemotherapeutics), the recruited CD8 T cells might not have sufficient antigen cross-presentation and cross-priming mediated by DCs and macrophages [8, 11]. Future studies are needed to investigate the optimal combination therapy by HIFU thermal ablation or hyperthermia with ICIs and adjuvants/chemotherapeutics to achieve systemic, long-term effects for cancer treatment.High-Intensity Focused Ultrasound Mechanical Ablation-Enhanced Immune Checkpoint Inhibitor TherapyHIFU mechanical ablation utilizes short pulse lengths (microsecond to millisecond) and low duty cycles to produce mechanical ablation of tissues while limiting tissue temperature increase. The primary physical mechanism of HIFU mechanical ablation is cavitation, which is defined as the formation, oscillation, and collapse of bubbles in the acoustic field. Cavitation can induce tissue damage by various mechanisms, including microjecting, streaming, and shear stresses [37]. The formation of cavitation in tissue by HIFU can be facilitated by the injection of exogenously made cavitation nuclei, for example, microbubbles or phase-changing materials (e.g., perfluorocarbon). Without the injection of cavitation nuclei, cavitation can be initiated using ultrasound pulses with high tensile pressure, which stretches the tissue and generates cavitation bubbles in situ. When extremely high tensile pressures are generated, HIFU can lead to complete liquefaction of the tumor tissue into submicron fragments, which is named histotripsy [38]. Several reports have shown that HIFU mechanical ablation can cause immunogenic cell death and release tumor debris in situ, promote antigen presentation, and enhance the inflammatory response [33].The clinical applications of ICIs in brain tumors (e.g., glioblastoma and neuroblastoma) have been challenging, potentially because these tumors harbor a “cold” immune microenvironment that lacks requisite T cells and sufficient TAAs and contains high densities of immunosuppressive cell populations [39, 40]. One recent study demonstrated that HIFU mechanical ablation combined with silica microshells mechanically disrupted glioblastoma tumors and augmented the efficacy of αPD-1 [13]. The combination of HIFU mechanical ablation with microshells and αPD-1 increased tumor-infiltrating CD8 and IFN-γ+CD8 T cells, prolonged tumor-free survival and protected against tumor rechallenge, suggesting the formation of long-term immune memory against glioblastoma. In a murine neuroblastoma model, Eranki et al. demonstrated that histotripsy potentially transformed immunologically “cold” tumors into responsive “hot’ tumors and provided an efficacious adjuvant to ICI therapy [14]. Histotripsy followed by systemic injection of αCTLA-4 and αPD-1 induced significant increases in intratumoral CD4, CD8α, and CD8α+ DCs in regional lymph nodes and circulating IFN-γ and decreases in circulating IL-10. Notably, the combination therapy improved long-term survival, achieved complete bilateral tumor regression, and induced an effective long-term immune memory response to suppress subsequent tumor engraftment. Other recent studies found that histotripsy stimulated more potent intratumoral CD8 T cells and antigen presentation than HIFU thermal ablation in a murine breast cancer model [15] and melanoma model [16]. One recent study showed that combining histotripsy with intratumor anti-CD40 agonist antibody, αCTLA-4, and anti-PD-L1 antibody (αPD-L1) significantly improved the therapeutic efficacy against ICI refractory murine melanoma [17].These findings [13–16] suggest that HIFU mechanical ablation alone, without the need for adjuvants, is sufficient to enhance ICI therapy for the treatment of cancers that are unresponsive to ICIs. One advantage of HIFU mechanical ablation over HIFU thermal ablation is that tumor fragmentation instead of tumor coagulation may protect TAAs and DAMPs from protein denaturation by excessive heat and stimulate more effective antitumor immune responses [15, 16, 33, 41].Ultrasound-Targeted Microbubble Destruction-Enhanced Immune Checkpoint Inhibitor TherapyThere is no consensus regarding the definition of low-intensity focused ultrasound (LIFU). It can be regarded as FUS with pulse intensity similar to that of diagnostic ultrasound. Microbubbles are made of a phospholipid, surfactant, albumin, or synthetic polymer shell filled with a high molecular weight gas with low water solubility. These microbubbles were initially introduced into the clinic as ultrasound contrast agents to enhance ultrasound signals from the blood circulation [42]. Over the past decades, they have been developed into theranostic agents. Their shells can be used for disease-specific targeting and loaded with drugs as carriers for controlled drug release at the LIFU-targeted region. Moreover, microbubble cavitation upon LIFU sonication can generate mechanical forces on surrounding tissue and induce vascular disruption [43].PD1/PD-L1 ICIs have been used in the clinic for the treatment of NSCLC in combination with chemotherapeutic drugs. However, the combination of these drugs leads to aggravated cardiotoxicity, hematotoxicity, hepatotoxicity, and neurotoxicity [44]. Li et al. used microbubbles as carriers of immunotherapy and chemotherapy drugs to produce antitumor effects while reducing the toxicities of the drug combination [18]. Docetaxel was loaded inside the lipid shell of the microbubbles, and αPD-L1 was conjugated to the surface of the microbubbles. UTMD improved drug delivery to the tumor potentially through three combined effects: αPD-L1 on the surface of the microbubbles specifically targeted the tumor cells; ultrasound sonication ruptured the microbubbles and released the carried drug at the LIFU-targeted tumor site; microbubble cavitation increased tumor permeability and promoted drug penetration across the vessel and into the tumor tissue. As a result, this therapeutic strategy inhibited tumor growth and improved the survival of mice implanted with tumor cells in the lung. It is worth to point out that lung diseases are often considered difficult to treat with FUS because the lungs are air-filled cavities. However, clinical studies have combined ultrasound and microbubbles to enhance drug delivery to the lungs of patients with pneumonia, acute respiratory distress syndrome, and NSCLC [45, 46]. It was proposed that because the diseased areas of the lung are filled with fluid, ultrasound waves could penetrate through the diseased area and leave normal air-filled areas of lung unaffected.Microbubbles were also used as “anti-vascular” agents to disrupt blood vessels and increase the antitumor effects of ICI therapy of colorectal cancer in a study by Bulner et al. [19]. They found that UTMD alone induced an instant shutdown of blood flow within tumor tissue and resulted in tumor necrosis in a mouse model of colorectal cancer. The combination of UTMD and αPD-1 treatment conferred better tumor growth constriction and a higher survival rate than USMB or αPD-1 alone and rejected subsequent tumor rechallenge. However, the results did not support that the combined UTMD and αPD-1 treatment shifted T-cell subpopulations to a more favorable antitumor state.In a murine breast cancer model, UTMD produced triple antitumor effects simultaneously: carrying an anti-CD326 antibody to target tumor cells, nonviral gene transduction of IFN-β by sonoporation, and tumor debulking by mechanical forces [20]. Such proximity of microbubbles to tumor cells using targeted microbubbles was crucial for effective sonoporation to transfect tumor cells. IFN-β expression plus αPD-1 led to a decreased tumor cell population and increased tumor-infiltrating CD8 T cells. The complete combination treatment attained greater tumor growth reduction in treated and distant tumors and prolonged survival than any partial treatments in the murine breast cancer model.Sonodynamic Therapy-Enhanced Immune Checkpoint Inhibitor TherapySDT utilizes LIFU to activate sonosensitizers and induces cytotoxicity [47, 48]. Unlike chemotherapy drugs that have massive toxicity on healthy cells, SDT induces tumor cell disruption only at the LIFU-targeted site. Preclinical studies have found that tumor cell debris generated by SDT could provide TAAs for initiating antitumor immunological effects [47, 48]. One report employed SDT using liposomes loaded with sonosensitizers and adjuvants [21]. Strikingly, SDT combined with αPD-L1 eradicated the primary tumor, suppressed distant tumor growth, inhibited whole-body metastasis in murine breast cancer models and produced sufficient immune memory responses to reject subsequent tumor rechallenge in murine breast and colorectal cancer models. The SDT-elicited antitumor effects, immune adjuvant-containing sonosensitizers, and αPD-L1-mediated systemic antitumor immune response were attributed to the robust antitumor response.Recently, Um et al. used nanobubbles loaded with a sonosensitizer (chlorin e6) for the treatment of pulmonary metastasis of colorectal cancer [22]. Upon sonication, these nanobubbles caused cell membrane disruption by cavitation, which triggers immunogenic cancer cell death and releases intact DAMPs for in situ cancer vaccination. The combination of αPD-L1 with nanobubbles loaded with the sonosensitizer effectively suppressed primary and metastatic tumors, which suggested that physically induced tumor cell death by the nanobubbles combined with SDT can augment the efficacy of ICIs. More work is needed to determine whether this strategy can improve long-term survival and generate long-lasting immune memory responses against tumor recurrence.DiscussionRecent publications have presented exciting and promising results that FUS modalities can improve ICI therapy. Combination therapies were reported to suppress tumor growth, achieve tumor remission, improve long-term survival, and prevent tumor recurrence for cancer types that are not readily responsive to ICI treatments. The field of FUS-enhanced IC therapy is still in its infancy, with all existing studies focused on proofing the concept. Further development of the combination strategy requires a multidisciplinary approach with a proper choice of FUS parameters for particular tumors, a complete examination of the correlation between FUS parameters and antitumor immune effects, a thorough evaluation of the biological mechanisms for therapeutic outcome, and a good understanding of the clinical challenges in cancer immunotherapy.Although each FUS modality has the capability to improve ICI immunotherapy, it is still unknown which regimen has the greatest potential to combine with ICIs. One major challenge is the inconsistent reporting of FUS parameters and antitumor immune effects, which prevents correlating FUS parameters with antitumor immune effects. It is important to standardize reporting on FUS procedures to include all key parameters, such as ultrasound frequency, intensity, pressure, duty cycle, pulse repetition frequency, sonication target locations, and sonication duration. It is also critical to establish standards in reporting antitumor immune effects to enable comparisons across different studies. Another challenge is that the choice of the optimal FUS modality to improve ICI immunotherapy may depend on tumor type.The biological mechanisms of each FUS-enhanced ICI therapy remain to be revealed. The reported HIFU thermal ablation-enhanced therapy required adjuvants to provide sufficient antigen cross-presentation and cross-priming for CD8 T cells. In contrast, HIFU mechanical ablation alone was sufficient to effectively stimulate antitumor immune responses to enhance ICI therapy. There was only one report on HIFU hyperthermia-enhanced ICI therapy. UTMD has great potential to improve ICI therapy through targeted and controlled release of therapeutics, sonoporation, and mechanical disruption of the blood vessels and tumor tissue. SDT induces tumor cell disruption only at the FUS-targeted site, resulting in reduced toxicity. Further investigations are warrant to better understand the biological mechanisms of each combination therapy.FUS-enhanced ICI therapies have already undergone early stage clinical evaluations. Currently, two clinical trials have begun to evaluate the combination of HIFU thermal ablation with pembrolizumab (αPD-1) for the treatment of various advanced solid tumors, such as melanoma, breast cancer, and Merkel cell carcinoma (ClinicalTrials.gov Identifier: NCT04116320 and NCT03237572). The primary outcome will assess a change in the CD8/CD4 T cell ratio in the ablation zone, and the secondary outcome will assess adverse events. Meanwhile, one clinical trial has started evaluating the use of UTMD to enhance the permeability of the blood-brain barrier without causing vascular damage to facilitate the delivery of nivolumab (αPD-1) to melanoma metastases in the brain and boost immunity in the brain (NCT04021420).For the successful clinical translation of FUS-enhanced ICI therapy, we need strong collaboration between ultrasound engineers and immunologists. Ultrasound engineers can optimize FUS parameters to induce the optimal biological effects that effectively induce antitumor immune responses with minimized side effects. Immunologists can better characterize the resulting antitumor immune responses and therapeutic outcomes. Through appropriate tuning of FUS exposure conditions and comprehensive immunological characterization, the prospect of unmasking the utility of FUS with ICI therapy could be attainable.Author ContributionsHC conceived the outline of the review article and edited the manuscript. 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Download referencesAuthor informationAuthors and AffiliationsDepartment of Genetic Engineering, Sungkyunkwan University, Suwon, 16419, Republic of KoreaSungrae Cho, Hocheol Shin, Kangsan Roh, Seungchan Cho, Eui-joon Kil, Hee-seong Byun, Sang-ho Cho & Sukchan LeeYeom Chang-Hwan hospital, Seoul, 06605, Republic of KoreaJin Sung Chae & Chang-Hwan YeomDepartment of Applied Chemistry, Dongduk Women’s University, Seoul, 02748, Republic of KoreaYujeong Shin, Haeun Song & Seyeon ParkDepartment of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul, 06351, Republic of KoreaYoungwook KimColorectal Cancer Branch, Division of Translational and Clinical Research, Research Institute, National Cancer Center, Goyang, 10408, Republic of KoreaByong Chul YooAuthorsSungrae ChoView author publicationsYou can also search for this author in
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PubMed Google ScholarContributionsS.R.C., C.Y. and S.L. designed the experiment and concepts. S.R.C. performed the experiments and analysis the data with J.C. The manuscript was drafted by S.R.C., H.S. assisted the experiments and Y.S. and H.S. performed HPLC experiments and analysis. Y.K. performed clustering of the cell viability data and AUC curve analysis. B.C.Y. distributed colorectal cancer cells. K.R., S.C.C., E.K., H.B., S.H.C., S.P., analysis and interpretation of data. All the authors discussed about the results and commented on manuscript.Corresponding authorsCorrespondence to
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Reprints and PermissionsAbout this articleCite this articleCho, S., Chae, J.S., Shin, H. et al. Hormetic dose response to L-ascorbic acid as an anti-cancer drug in colorectal cancer cell lines according to SVCT-2 expression.
Sci Rep 8, 11372 (2018). https://doi.org/10.1038/s41598-018-29386-7Download citationReceived: 30 January 2018Accepted: 10 July 2018Published: 27 July 2018DOI: https://doi.org/10.1038/s41598-018-29386-7Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard
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