Nicotine promotes epithelial to mesenchymal transition and gemcitabine resistance via hENT1/RRM1 signalling in pancreatic cancer and chemosensitizing effects of Embelin-a naturally occurring benzoquinone.

Pancreatic cancer is lethal due to poor prognosis with 5-year survival rate lesser than 5 %. Gemcitabine is currently used to treat pancreatic cancer and development of chemoresistance is a major obstacle to overcome pancreatic cancer. Nicotine is a known inducer of drug resistance in pancreatic tumor micro-environment. Present study evaluates chemoresistance triggered by nicotine while treating with gemcitabine and chemosensitization using Embelin. Embelin is a naturally occurring benzoquinone from Embelia ribes possessing therapeutic potency. To develop nicotine-induced chemo-resistance, pancreatic cancer cells PANC-1 and MIA PaCa-2 were continuously treated with nicotine followed by exposure to gemcitabine. Gemcitabine sensitivity assay and immunoblotting was performed to assess the chemo-resistance. Antiproliferative assays such as migration assay, clonogenic assay, Mitochondrial Membrane Potential (MMP) assay, dual staining assay, comet assay, Reactive Oxygen Species (ROS) assay, cell cycle analysis and immunoblotting assays were performed to witness the protein expression involved in chemoresistance and chemosensitization. Epithelial to mesenchymal transition was observed in nicotine induced chemoresistant cells. Gemcitabine sensitivity assay revealed that relative resistance was increased to 6.26 (p < 0.0001) and 6.45 (p < 0.0001) folds in resistant PANC-1 and MIA PaCa-2 compared to parental cells. Protein expression studies confirmed resistance markers like hENT1 and dCK were downregulated with subsequent increase in RRM1 expression in resistant cells. Embelin considerably decreased the cell viability with an IC50 value of 4.03 ± 0.08 µM in resistant PANC-1 and 2.11 ± 0.04 µM in resistant MIA PaCa-2. Cell cycle analysis showed Embelin treatment caused cell cycle arrest at S phase in resistant PANC-1 cells; in resistant MIA PaCa-2 cells there was an escalation in the Sub G1. Embelin upregulated Bax, γH2AX, p53, ERK1/2 and hENT1 expression with concomitant down regulation of Bcl-2 and RRM1. Bioactive molecule embelin, its combination with gemcitabine could provide new vistas to overcome chemo resistance in pancreatic cancer.

RRM2 Regulates Sensitivity to Sunitinib and PD-1 Blockade in Renal Cancer by Stabilizing ANXA1 and Activating the AKT Pathway.

Renal cell carcinoma (RCC) is a malignant tumor of the kidneys. Approximately 70% of RCC cases are clear cell renal cell carcinoma with von Hippel-Lindau (VHL) gene mutation and activation of the vascular endothelial growth factor (VEGF) pathway. Tyrosine kinase inhibitors (TKIs) targeting VEGF have emerged as promising agents for RCC treatment. Apart from primary resistance, acquired resistance to TKIs after initial tumor regression is common in RCC. Recently, immune checkpoint inhibition, including PD-1/PD-L1 blockade, alone or in combination with TKIs has improved the overall survival of patients with RCC. Ribonucleotide reductase subunit M2 (RRM2) has been reported in many types of cancer and has been implicated in tumor progression. However, the role of RRM2 in TKIs resistance in RCC remains unclear. In this study, the authors have demonstrated that RRM2 is upregulated in sunitinib-resistant RCC cells and patient tissues. They also find that RRM2 stabilizes ANXA1 and activates the AKT pathway independent of its ribonucleotide reductase activity, promoting sunitinib resistance in RCC. Moreover, RRM2 regulated antitumor immune responses, and knockdown of RRM2 enhance the anti-tumor efficiency of PD-1 blockade in renal cancer. Collectively, these results suggest that aberrantly expressed RRM2 may be a promising therapeutic target for RCC.

A genotype 2b NS5B polymerase with novel substitutions supports replication of a chimeric HCV 1b:2b replicon containing a genotype 1b NS3-5A background.

HCV diversity suggests that evaluation of HCV inhibitors for broad genotypic efficacy is warranted. The replicon system enables cell-culture compound efficacy evaluation against an active replication complex, and a functional replicon dependent upon a genotype 2b polymerase would augment existing cell-culture efficacy studies that are presently limited to genotype 1a, 1b, and 2a replicons. We made a chimeric Neo(r) 1b:2b replicon where genotype 2b NS5B was inserted into a genotype 1b NS3-5A background and transfected replicon RNA to generate Neo(r) cell lines. All cell lines contained novel substitutions within NS5B which were subsequently engineered into the parental 1b:2b replicon and shown to enhance replication to various degrees. A single NS5B M31I substitution enhanced replication to levels sufficiently robust to quantify sensitivity to HCV inhibitors in a transient replication assay. The M31I 1b:2b replicon was similarly sensitive to an active-site nucleoside inhibitor of NS5B as genotype 1b replicons, but was insensitive to two non-nucleoside inhibitors which were otherwise efficacious against the genotype 1b replicons. This work describes a novel HCV replicon sustained by a genotype 2b polymerase that is sufficiently robust for quantifiable analysis in a transient replication assay, and demonstrates its utility in characterizing anti-HCV compounds for cross-genotypic efficacy.