key: cord-0864396-clvnwy1l authors: Lu, Haofeng; Zhou, Lin; Zuo, Hongping; Le, Wenjin; Hu, Jianfei; Zhang, Tiequan; Li, Mi; Yuan, Yufeng title: Ivermectin synergizes sorafenib in hepatocellular carcinoma via targeting multiple oncogenic pathways date: 2022-05-14 journal: Pharmacol Res Perspect DOI: 10.1002/prp2.954 sha: c7730187cc6447f610c8f4789efdf5b668eaa840 doc_id: 864396 cord_uid: clvnwy1l Advanced hepatocellular carcinoma (HCC) results in generally poor clinical outcomes and necessitates better therapeutic strategies. Ivermectin, which is an existing anti‐parasitic drug, has been recently identified as a novel anti‐cancer drug. In line with previous efforts, this work demonstrates the translational potential of ivermectin to treat advanced HCC. We demonstrated that ivermectin at clinically relevant concentrations was active against growth and survival in multiple HCC cell lines. We showed that ivermectin had the potential to inhibit metastasis and target HCC stem cell functions. Mechanism studies correlated well with cellular phenotypes observed in ivermectin‐treated cells, and demonstrated inhibition of mTOR/STAT3 pathway, suppression of epithelial mesenchymal transition (EMT) and reduced expression of stem cell markers. We further demonstrated that ivermectin inhibited tumor formation and growth in HCC xenograft mouse model, without causing significant toxicity in the mice. Using combination index (CI), we showed that ivermectin and sorafenib were synergistic in HCC in vitro, and this was further confirmed in vivo. Our work demonstrates the potent anti‐HCC activities of ivermectin and its multiple targets on essential oncogenic pathways. Our findings provide preclinical evidence to initialize clinical trial using ivermectin and sorafenib for treating advanced HCC. Hepatocellular carcinoma (HCC) is the third most frequent cause of cancer-related deaths worldwide, and its incidence continues to grow. 1 Standard of care for HCC patients include surgery, transarterial chemoembolization, radiotherapy and targeted therapy. However, most patients are diagnosed at their late stages and hence are resistant to chemotherapy and not indicative for surgery. 2, 3 Sorafenib, an oral multikinase inhibitor, is routinely used for the treatment of advanced HCC. 4 However, clinical evidence shows that sorafenib only prolongs median survival and time to progression by 3 months. Patients typically develop sorafenib resistance leading to disease relapse. 5 Therapeutic strategies that augment sorafenib efficacy may aid to circumvent resistance. In addition, drug repurposing can facilitate rapid clinical translation due to known pharmacological and pharmaceutical profilings. Ivermectin is a FDA-approved anti-parasitic drug and widely used to treat infections of onchocerciasis and gastrointestinal parasites, with mild and self-limiting adverse effects. 6 Recently, increasing evidence highlights that ivermectin is a novel anticancer drug. 7 Ivermectin demonstrates anti-proliferative and pro-apoptotic activities in a variety of tumors, including ovarian cancer, renal cell carcinoma, glioblastoma and hematological malignancies. [8] [9] [10] [11] Apart from tumor bulk/differentiated cells, ivermectin also inhibits cancer stem-like cells and tumor angiogenesis. 12, 13 In addition, ivermectin augments the effects of anti-cancer agents and even reverses the drug resistance in cancer cells. [14] [15] [16] We hypothesized that ivermectin could be used to overcome sorafenib resistance in HCC. In this pre-clinical study, we investigated the potential of ivermectin to treat advanced HCC by addressing the following questions: After drug treatment, proliferation and apoptosis were measured using BrdU (Bromodeoxyuridine/5-bromo-2'-deoxyuridine) Cell Proliferation Assay Kit (Abcam) and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) Assay Kit (R&D Systems) according to their manufacturer's instructions. The absorbance was measured at microplate reader (Thermo Fisher Scientific). The concentrations of single drug IC 50 were first determined using proliferation assay. The cells were then treated with increasing doses of either ivermectin or sorafenib, or an equipotent constantratio combination of both drugs, followed by proliferation measurement. The CI values were calculated and plotted using CompuSyn. exe based on dose and effect of single drug alone, and drug combinations to determine if these were synergistic (CI < 0.9), additive (CI 0.9-1.1) or antagonistic (CI > 1.1). 1000 cells together with drugs were suspended in noble agar were sacrificed using CO 2 and followed by cervical dislocation. Results were obtained from at least three time-independent experiments and reported as means with standard error. Statistical analyses of the differences between two groups were performed using one-way analysis of variance (ANOVA) and unpaired Student's t test. The PRISM statistical software (version 9.0, GraphPad Inc.) was used in analyzing all statistical comparisons. A p < 0.05 was considered statistically significant in all cases. To evaluate the efficacy of ivermectin in HCC, we had performed both proliferation and apoptosis assays on HCC cells after treatment with clinically achievable concentrations of ivermectin. 17 Using three HCC To determine whether ivermectin has potential to affect HCC migration, we performed transwell assay using serum as chemoattractant. We found that ivermectin at concentrations ranging from 2.5 to 10 μM decreased HCC migrations by up to 50% ( Figure 1C and D). To examine subpopulations within a cell line with stem cell-like properties, we conducted anchorage-independent colony formation assay. 18 Ivermectin potently inhibited HCC anchorage-independent colony formation ( Figure 1E and F), suggesting the inhibitory effect of ivermectin in HCC stem cells. Comparing with inhibition of migration and survival, we noted that ivermectin seemed to be more effective in targeting HCC growth and colony formation. To determine the underlying mechanisms of ivermectin's action, we examined mTOR/STAT3 pathway in HCC cells exposed to ivermectin because (1) ivermectin inhibits mTOR pathway in ovarian cancer 16 ; (2) mTOR activates STAT3 in cancer cells; (3) mTOR/STAT3 critically regulates cancer cell growth and survival. 19 We also examined other pathways focusing on essential molecules that have critical roles in promoting migration and stemness, respectively. Consistent with the recent report, 16 we found that ivermectin decreased mTOR phosphorylation at Ser2448 (Figure 2A) . We further observed the decreased STAT3 phosphorylation at both Ser727 and Tyr705. Tyrosine phosphorylation at 705 leads to STAT3 nuclear translocation and activation of gene transcription. 20 Consistently, we observed the de- Given the findings above, we next evaluated the efficacy of ivermectin in HCC in vivo using xenograft mouse model. Mice were inoculated with SNU-182 cells via subcutaneous injection to one flank site. We analyzed the effects of ivermectin on HCC formation as well as progression. We also weighed the mice and monitored for signs of possible toxicity throughout the whole duration of drug treatment. Compared with vehicle control, there were no significant differences on mice body weight in drug-treated group up to 9 days treatment of 10 mg/kg ivermectin ( Figure 3A ). Approximate 20%, 60% and 90% mice in control group whereas 0%, ~15% and ~20% in ivermectin-treated group developed palpable tumor at 3, 6 and 9 days post-inoculation ( Figure 3B ), demonstrating that ivermectin significantly suppresses HCC tumor formation. To evaluate the effect of ivermectin in HCC growth, we only initialized ivermectin treatment after tumor formation. When tumor reached ~150 mm 3 , mice were randomly divided into three groups receiving vehicle, oral ivermectin at two different doses. For up to 24 days of drug treatment, there were no significant differences in body weight and other features (e.g., skin, fur and motion) between control and mice group receiving 10 mg/kg ivermectin ( Figure 3C ). We found that both 5 and 10 mg/kg of ivermectin significantly decreased tumor size, and this reduction was dose-dependent ( Figure 3D ). These indicate that ivermectin at non-toxic doses effectively inhibits HCC tumor growth in mice in a dose-dependent manner. Combination therapy is often used in cancer patients to improve the probability of therapeutic responses and decrease the likelihood of acquired resistance. 23 To investigate the translational potential of ivermectin in HCC, it is therefore important to determine the combinatory effects of ivermectin with standard of care drug for advanced HCC, such as sorafenib. 5 Combination index (CI) provides a quantitative measure of the extent of drug interaction via estimating dose-effect data from single and combined drug treatments. 24 We performed combination studies using ivermectin and sorafenib, and calculated CI using proliferation assay. Our fraction-CI analysis indicated that CI value were less than 1 between ivermectin and sorafenib in all tested HCC cell lines (Figure 4) , demonstrating that ivermectin and sorafenib combination is synergistic in HCC cells. We further confirmed synergism of the combination in vivo using HCC xenograft mouse model. The combination of ivermectin and sorafenib was significantly more effective to decrease HCC tumor Drug repurposing, or the use of currently approved drugs for new indications, is a plausible alternative strategy to uncover anti-cancer drugs. 25, 26 To identify approved drugs with anti-HCC activity, we screened a library of antimicrobials and antihelminthics on multiple HCC cell lines using CellTiter-Glo viability assay. Here, we report that ivermectin, an anthelminthic drug, is an attractive candidate for HCC treatment. Our findings demonstrate that ivermectin inhibits growth, survival, migration and anchorage-independent colony formation of HCC cells. It acts synergistically with sorafenib in vitro and in vivo, via suppressing multiple essential oncogenic pathways. Importantly, the effective doses of ivermectin in HCC is clinically achievable without causing toxicity in mice. Consistent with many studies on the inhibitory effects of ivermectin on cancer, [8] [9] [10] [11] we show that ivermectin is effective against HCC. Using multiple HCC cell lines that represent different tumor cellular origins and genetic profiles, we showed that ivermectin inhibited growth and induced apoptosis for all tested HCC cell lines ( Figure 1A and B) . Mandy et al. have tested the effects of ivermectin on 28 cancer cell lines covering many types of cancer and found that IC 50 of the most sensitive lines to ivermectin was ~5 μM. 17 The IC 50 (based on proliferation assay) of ivermectin in HCC is at ~2.5 to 5 μM, suggesting that HCC is more sensitive to ivermectin compared to other cancers. Pharmacokinetic data in humans has shown that 5.2 μM of ivermectin is detected in healthy subjects with a dose of 2 mg/kg, 27 28 Our work also demonstrated that ivermectin targeted stem-like cells in HCC and this cell subset was more sensitive to ivermectin than bulk HCC cells as shown by anchorage-independent cell growth assay ( Figure 1E and F). ivermectin is an inhibitor of cancer stem-like cells and restricts cancer stem cell formation, 13, 29 which is also consistent with our in vivo finding that ivermectin inhibited HCC tumor formation ( Figure 3B ). Using xenograft mouse model, we further demonstrated that ivermectin significantly inhibited HCC growth ( Figure 3C and D) . The doses of ivermectin used in our animal model were equivalent to 0.7-1.4 mg/kg in humans, which was a dose below the highest dose safely used in human. 27 Continuous high-dose ivermectin appears to be safe in patients. 30 This is consistent with our findings that we did not observe any signs of toxicity in ivermectin-treated mice ( Figures 3A, C and 5A) . Ivermectin has been shown to augment efficacy of anti-cancer agents, such as docetaxel, cyclophosphamide and tamoxifen. 17 Our combination index of ivermectin and sorafenib was all less than 1, clearly indicating that the combination is synergistic (Figure 4) . The synergy between ivermectin and sorafenib, which was further shown in vivo ( Figure 5B) , is critical to support clinical trial testing of sorafenib and ivermectin combination in HCC. Our mechanism studies demonstrated that ivermectin inhib- All authors report no conflict of interest. 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