Minnelide Overcomes Oxaliplatin Resistance by Downregulating the DNA Repair Pathway in Pancreatic Cancer

Abstract

Introduction: Oxaliplatin is part of pancreatic cancer therapy in the FOLFIRINOX or GEMOX/XELOX regimen. DNA damage repair is one of the factors responsible for oxaliplatin resistance that eventually develops in this cancer. Triptolide/Minnelide has been shown to be effective against pancreatic cancer in preclinical trials. In this study, we evaluated the efficacy of combination of triptolide and oxaliplatin against pancreatic cancer.

Methods: Highly aggressive pancreatic cancer cells (MIA PaCa-2 and PANC-1) were treated with oxaliplatin (0-10 μM), low-dose triptolide (50 nM), or a combination of both for 24-48 h. Cell viability, apoptosis, and DNA damage were evaluated by appropriate methods. Nucleotide excision repair pathway components were quantitated using qPCR and Western blot. Combination of low doses of Minnelide and oxaliplatin was tested in an orthotopic murine model of pancreatic cancer.

Results: Proliferation of pancreatic cancer cells was markedly inhibited by combination treatment. Triptolide potentiated apoptotic cell death induced by oxaliplatin and sensitized cancer cells towards oxaliplatin-induced DNA damage by suppressing the oxaliplatin-induced DNA damage repair pathway. Combination of low doses of Minnelide and oxaliplatin inhibited tumor progression by inducing significant apoptotic cell death in these tumors.

Conclusions: Combination of low doses of Minnelide and oxaliplatin has immense potential to emerge as a novel therapeutic strategy against pancreatic cancer.

Keywords: DNA damage pancreatic cancer; Minnelide; NER; Oxaliplatin; Triptolide.

Figure 1

TPL and oxaliplatin synergize in decreasing viability of pancreatic cancer cell lines MIA PaCa-2 and PANC-1. MIA PaCa-2 (a) and PANC-1 (b) cells were incubated with TPL (50 nM) and/or oxaliplatin (5μM) for 48 hours. Normalized isobologram for triptolide and oxaliplatin in the MIA PaCa-2 (c) and PANC-1 (d) cell lines. Data points located at lower left inside the triangle indicate synergism. Fractional affected-combination index (Fa-CI) plot in combination index analysis in the MIA PaCa-2 (e) and PANC-1 (f) cell lines. Combination index values <1, =1 or >1 indicate synergism, additivity and antagonism respectively. Data are mean ± SEM, n=3, *p<0.05.

Figure 2

TPL enhances oxaliplatin induced apoptosis in pancreatic cancer cell lines MIA PaCa-2 and PANC-1. MIA PaCa-2 and PANC-1 cells were incubated with TPL (50 nM) and/or oxaliplatin (4 μM) for 24 hours. TPL (200 nM) was used a positive control. Cleaved Caspase-3 levels in MIA PaCa-2 cells (a) and PANC-1 cells (b). Cleaved PARP levels in MIA PaCa-2 cells (c) and PANC-1 cells (d). Data are mean ± SEM, n=3, *p<0.05 compared to control and each drug alone.

Figure 3

Combination therapy with TPL and oxaliplatin increases DNA damage. MIA PaCa-2 and PANC-1 were incubated with TPL (50 nM) and oxaliplatin (4 μM) for 24 hours. SN-38 was used as a positive control. γH2A.X levels in MIA PaCa-2 (a) and PANC-1 (b) cancer cells. Data are mean ± SEM, n=3, *p<0.05 compared to control and each drug alone.

Figure 4

TPL abrogates oxaliplatin induced mRNA expression of various components of Nucleotide Excision Repair (NER) pathway. MIA PaCa-2 (a, b) and PANC-1 (c, d) pancreatic cancer cells were incubated with TPL (50 nM) and/or oxaliplatin (4 μM) for 24 hours. mRNA levels of ERCC1, XPA, XPB, XPC, XPD, XPF and XPG were tested in MIA PaCa-2 and PANC-1 cancer cells. Data are mean ± SEM, n=3. *p<0.05 compared to control and oxaliplatin.

Figure 5

Oxaliplatin increases the protein expression of key components of NER pathway while triptolide decreases it. Effect of TPL (50 nM) and/or oxaliplatin (4 μM) on the protein levels of XPA, XPB, XPC, ERCC1, XPD and XPF in MIA PaCa-2 (a) and PANC-1 (b) cancer cells.

Figure 6

TPL abrogates oxaliplatin induced upregulation of AP1 promoter activity. Effect of TPL (50 nM) and/or oxaliplatin (4 μM) on AP1 promoter activity in MIA PaCa-2 (a) and PANC-1 (b) pancreatic cancer cells. Phorbol 12-Myristate 13-Acetate (PMA: 10ng/ml) was used as a positive control. Data are mean ± SEM, n=4. *p<0.05 compared to control and oxaliplatin.

Figure 7

Combination treatment of Minnelide and oxaliplatin suppressed tumor growth in vivo by inducing apoptotic cell death in tumor cells. MIA PaCa-2 cells (1 × 10⁵) were injected into the tail of pancreas of 4-6 weeks old athymic nude mice (7 mice /group). Animals were randomized into 4 groups and treatment was started 3 weeks after surgery: saline with 50 μl DMSO, Minnelide 0.15 mg/kg/day, oxaliplatin 6 mg/kg/week and Minnelide combined with oxaliplatin. To assess the toxicity profile, body weight was plotted at start and end of treatment (c). At day 33, the animals were euthanized and tumors weight (a) and volume (b) were documented. Fixed tissue in 10 % neutral buffered formalin was used for TUNEL staining later (d and e). Data are mean ± SEM, n=7. *p<0.05 compared to all other groups.

Figure 8

Schematic diagram showing the inhibitory effects of triptolide on oxaliplatin induced upregulation of nucleotide excision repair pathway. Triptolide inhibits AP1 driven transcription of various genes of the NER pathway.