Blockade of Uttroside B-Induced Autophagic Pro-Survival Signals Augments Its Chemotherapeutic Efficacy Against Hepatocellular Carcinoma

Abstract

Our previous study has demonstrated that Uttroside B (Utt-B), a saponin isolated from the leaves of Solanum nigrum Linn induces apoptosis in hepatic cancer cells and exhibits a remarkable growth inhibition of Hepatocellular Carcinoma (HCC). Our innovation has been granted a patent from the US (US 2019/0160088A1), Canada (3,026,426.), Japan (JP2019520425) and South Korea (KR1020190008323) and the technology have been transferred commercially to Q Biomed, a leading US-based Biotech company. Recently, the compound received approval as ‘Orphan Drug’ against HCC from US FDA, which reveals the clinical relevance of evaluating its antitumor efficacy against HCC. In the present study, we report that Utt-B promotes pro-survival autophagy in hepatic cancer cells as evidenced by the increased expression of autophagy-related proteins, including LC3-II, Beclin1, ATG 5, and ATG 7, as well as a rise in the autophagic flux. Hence, we investigated whether Utt-B-induced autophagic response is complementing or contradicting its apoptotic program in HCC. Inhibition of autophagy using the pharmacological inhibitors, Bafilomycin A1(Baf A1), and 3-methyl adenine (3-MA), and the biological inhibitor, Beclin1 siRNA, significantly enhances the apoptosis of hepatic cancer cells and hence the cytotoxicity induced by Utt-B. We also found increased expression of autophagy markers in Utt-B-treated xenografts derived from HCC. We further analyzed whether the antimalarial drug, Chloroquine (Cqn), a well-known autophagy inhibitor, can enhance the anticancer effect of Utt-B against HCC. We found that inhibition of autophagy using Cqn significantly enhances the antitumor efficacy of Utt-B in vitro and in vivo, in NOD SCID mice bearing HCC xenografts. Taken together, our results suggest that the antitumor effect of Utt-B against HCC can be further enhanced by blocking autophagy. Furthermore, Utt-B in combination with Cqn, a clinically approved drug, if repurposed and used in a combinatorial regimen with Utt-B, can further improve the therapeutic efficacy of Utt-B against HCC.

Augmented Efficacy of Uttroside B over Sorafenib in a Murine Model of Human Hepatocellular Carcinoma

“…Our previous study screened cancer cells of various origins for their cytotoxicity towards Utt-B, wherein, the HCC cells exhibited remarkable cytotoxic potential [ 13 ]. Our very recent study has unraveled, with mechanism-based evidence, better modes to further improve the chemotherapeutic potential of Utt-B against HCC [ 9 ]. Except for a couple of in silico observations, there are no reports on the biological activity of Utt-B, other than ours, to date.…”

“…The current study depicts Utt-B as a better anti-HCC chemotherapeutic in comparison with sorafenib, the standard drug used to treat the disease. Our previous studies demonstrated the exceptional anti-HCC efficacy of Utt-B, which is a strong inducer of caspase-mediated apoptosis [ 9 , 13 ]. However, a comparative evaluation of the anti-HCC potency of Utt-B with sorafenib, the most studied US FDA-approved drug against HCC, was a requisite for better apprehension [ 18 ].…”

“…For instance, a recent study utilized a transcriptomics-based drug repurposing approach to comparatively evaluate the growth inhibitory effects of novel drugs against sorafenib-resistant HCC [ 25 ]. The human liver cancer cell line, HepG2 was used as a tool for the in vivo HCC model, due to its enhanced sensitivity towards both drugs, as per our previous observations [ 9 , 13 ]. Our data depict that Utt-B possesses credibility to function as a better anti-HCC drug than sorafenib.…”

“…Immunoblotting, clonogenic, and wound healing assays were performed as previously described [ 9 ]. Immunoblotting analysis was performed as described previously [ 13 ] and the quantification of the blots were carried out using ImageJ software.…”

“…Our discovery of Utt-B, a saponin derived from the plant black nightshade ( Solanum nigrum Linn), as a candidate drug against HCC, has attracted global recognition by gaining multi-national patents [USA (US2019/0160088A1), Canada (3,026,426), Japan (JP2019520425), Korea (KR1020190008323)], and commercial technology transfer (Q Biomed, Inc., New York, NY, USA). Our previous studies have revealed a complexity in Utt-B pharmacodynamics where we report that the drug induces an apoptotic mode of cell death in HCC cells but simultaneously functions as an inducer of autophagic signals, the inhibition of which further enhances the therapeutic index of Utt-B against HCC [ 9 ]. Importantly, Utt-B has now gained the ‘orphan drug’ status as designated by the U.S. FDA.…”