(W0930-04-28) Enhanced Cytotoxic Effect of Paclitaxel in Combination with Calcipotriol on Human Pancreatic Cancer Cells

Purpose: Pancreatic ductal adenocarcinoma (PDAC) continues to be as one of the leading causes of cancer-related mortality in the USA, with a 5-year overall survival rate of less than 10%. PDAC stroma functions as a barrier that affect drug delivery to tumor cells and leads to intrinsic chemoresistance. Quiescent human pancreatic stellate cells (HPSCs), a main part of stroma, store vitamin A as cytoplasmic lipid droplets, and when activated, they transform into myofibroblast-like cells characterized by the loss of vitamin A droplets. Activated HPSCs create growth-permissive tumor microenvironment. The activation of vitamin D receptors (VDR) may increase lipid droplets in HPSCs and convert the cells to their quiescent state which induces stromal remodeling and increase chemotherapy delivery to tumor cells. We hypothesized that the combination of calcipotriol, a vitamin D₃ analogue, with paclitaxel (PTX), a cancer cell-directed chemotherapy, acts in synergism to enhance PTX efficacy. Here, we studied the impact of calcipotriol on VDR and lipid droplets accumulation to show the potential merit of combination therapy on cancer cells in vitro.

Methods: Human pancreatic cancer cells (ASPC-1 and Miapaca-2) were used to compare the effect of combination therapy of PTX and calcipotriol with PTX alone. MTT cell viability assay was performed, and cells were treated with PTX alone or in combination with calcipotriol for 72 hours using vehicle as control. The cells were treated with a concentration of 2.5 nM of PTX and 2 µM, 5 µM, or 8 µM of calcipotriol, respectively. We used CompuSyn software for the assessment of drug combination index (CI). VDR protein expression levels were determined in ASPC-1 cells and HPSCs by western blot. Cells were treated with vehicle (1% DMSO), 100 nM, 1 µM, or 8 µM of calcipotriol for 72 hours. Cells lysates were obtained by treating the cells with lysis buffer and samples were run under reducing conditions on 10% polyacrylamide gels. Signal intensities were quantified by using image lab software and normalized to GAPDH by calculating VDR/GAPDH ratio. Exhibition of vitamin A-containing droplets in cultured HPSCs (fully activated cells) was visualized by oil-red staining. Cells cultured in 24-well plates were treated with vehicle, 100 nM, or 8 µM of calcipotriol and lipid droplets were visualized under light microscopy.

Results: PTX treatment of ASPC-1 and Miapaca-2 cells showed an IC50 of 25 nM and 9 nM, respectively (Figure 1A). The combination of PTX at a concentration of 2.5 nM with calcipotriol increased its cytotoxic effect. Combination therapy showed a significant decreased cell viability of ASPC-1 cells at calcipotriol concentrations of 5 µM (65.71%, P=0.0091) and 8 µM (57.09%, P=0.00253) when compared to PTX therapy alone (99.48%). It also showed a significant decreased cell viability of Miapaca-2 cells at calcipotriol concentrations of 2 µM (71.72%, P=0.00282), 5 µM (61.83%, P=0.00561), and 8 µM (36.36%, P=0.00027) when compared to PTX therapy alone (100%) (Figure 1B). The combination index plot against the fractional effect showed a synergistic effect in Miapaca-2 at fixed PTX concentration of 2.5 nM in combination with 2, 5, or 8 µM of calcipotriol, respectively (CI < 1). ASPC-1 cells showed synergism only at a concentration of 8 µM of calcipotriol (CI = 0.77) (Figure 1C). Calcipotriol increased VDR protein expression (normalized to VDR/GAPDH) in ASPC-1 cells in a dose dependent manner when compared to vehicle group (Figure 2). Increased VDR expression was also observed in HPSCs. Calcipotriol significantly increased the number of vitamin A lipid droplets in HPSCs cytoplasm at a concentration of 100 nM (average of 2.11 droplets per cell, P< 0.0001) when compared to vehicle (average of 0.35 droplets per cell). Calcipotriol also significantly increased lipid droplets at a concentration of 8 µM (average of 2.76 droplets per cell, P< 0.0001) when compared to vehicle and calcipotriol treatment of 100 nM (P=0.0064) (Figure 3).

Conclusion: Our data showed that the combination of calcipotriol with PTX is a potentially effective combination for the treatment of pancreatic cancer. Compared to single PTX treatment, combination therapy showed a synergistic and significant cytotoxic effect at low concentration of PTX (2.5 nM). We showed that VDR acts as a stromal regulator in pancreatic cancer and pancreatic stellate cells. VDR activation increased HPSCs cytoplasmic lipid droplets that may convert stellate cells to their quiescent state and may disrupt tumor-promoting activity of PDAC stroma to overcome chemotherapeutic drug resistance. The effect of calcipotriol and combination therapy on other in vitro fibroblastic biomarkers in HPSCs are currently investigated and preclinical in vivo studies are in progress to further explore the merit of this combination in appropriate PDAC animal models.

Figure 1. Effect of PTX single treatment on viability of ASPC-1 and Miapaca-2 cells (A). Cytotoxic effect of PTX treatment in combination with Calcipotriol compared to PTX single treatment (B). Combination Index (CI) plot and values using CompuSyn Software (C). CI>1: Antagonism, CI=1: Additive effect, and CI <1: Synergistic effect.

Figure 2. Effect of different concentrations of calcipotriol on VDR expression in ASPC-1 cells

Figure 3. Effect of calcipotriol on vitamin A lipid droplets in HPSCs using Oil-Red lipid assay. Lipid droplets are stained red, nuclei are stained blue, and visualized at x20 magnifications under light microscopy. Quantification of droplets was performed using ImageJ software.