Sensitizing Pancreatic Cancer Cells to Chemotherapeutics by Modulating Intracellular Iron Homeostasis

Currently, the only successful treatment for pancreatic cancer is invasive surgery. Previous studies to develop chemotherapeutic agents targeting pancreatic tumors have not led to any identifiable success. Due to the extreme vascularization of pancreatic tumors, we hypothesized that the overexpression of heme oxygenase‐1 (HO‐1) is responsible for the resistance of pancreatic cancer cells to chemotherapeutic drugs. Coinciding with elevated HO‐1expression, pancreatic cancer cells have increased expression of the heavy chain subunit of the intracellular iron‐storage protein ferritin (FTH‐1). To test our hypothesis, a series of experiments were designed to modulate the expression levels of HO‐‐1 and FTH‐‐1 in the human pancreatic cancer cell line Mia Paca‐2, to test effects on the cytotoxicity of the platinum based chemotherapeutic agent oxaliplatin. Reducing HO‐‐1 expression by small interfering RNA (siRNA) sensitized Mia Paca‐2 cells to oxaliplatin. Similarly, oxaliplatin induced more cell death in cancer cells with transiently and stably decreased FTH‐1 expression. Furthermore, inhibiting HO‐1 enzymatic activities by zinc protoporphyrin‐9 promoted cytotoxic activities of oxaliplatin. Our studies provide evidence that intracellular iron homeostasis is involved in the pancreatic tumor cells' responses to oxaliplatin. We are currently exploring the molecular mechanism of how modulating intracellular iron homeostasis sensitizes pancreatic tumor cells to chemotherapeutics. We expect these approaches will lead to a usable, therapeutic treatment for pancreatic cancer. Objectives Study whether reducing the expression of HO‐1 in pancreatic tumor cells improves the therapeutic effects of oxaliplatin. Investigate whether expression levels of FTH‐1 are involved in the pancreatic tumor cells' responses to oxaliplatin. Examine whether inhibiting HO‐1 enzymatic activities by zinc protoporphyrin‐9 sensitizes pancreatic cancer cells to oxaliplatin. Methods Cell line and incubation method For all of our experimentation, we used a human secondary cell line of Mia Paca‐2 cells. We chose this cell line because of its hardiness and resilience to many drugs already available on the common market. Furthermore, since our study involved the regulatory metabolism of iron, the expression of Heme Oxygenase‐1 and Ferritin Heavy Chain‐1 are naturally overexpressed in this cell line. For our experiments, the cells were seeded on 10‐cm plates in DMEM High Glucose Media with a 10% Fetal Bovine Serum along with a 5% solution of Pen Strep antibiotic. Cells were allowed to grow to 70–80% confluencey before being split and replated following the conditions above. The plated cells were incubated at 37° C and 5% CO 2 . Zinc Protoporphyrin‐IX Experiment Mia Paca‐2 cells were seeded in a confluencey of 9.0 × 10^5 cells per well in a 12‐well plate containing 1.5 mL of media. Cells were incubated overnight at 37° C and 5% CO 2 . Dry Zinc Protoporphyrin‐IX was weighed out and suspended in DMSO to reach the proper concentration measured in Molarity. The proper concentration was then added to each well containing media and plated cells. Western Blot Analysis Mia Paca‐2 cells were plated at a concentration of 9.0×10^5 cells per well in a 6‐well plate containing 3 mL of media. Cells were then incubated overnight at 37°C and 5% CO 2 . After incubation, the cells were subjected to a transient knockdown of either HO‐1 or FTH‐1 antibody in Opti‐MEM media with a Lypofectamine reagent. Once added, the cells were incubated overnight at 37° C and 5% CO 2 , again. After incubation, the cells were harvested and collected in 1.5 mL mictrocentrifuge tubes at a concentration of 1.0×10^6 cells. Once collected, these cells were then probed with 1X SDS loading buffer at a concentration of 5.0×10^3 cells/mL. Cells were then sonicated and denatured at 100°C for 5 minutes. Once denatured, the cells were spun down in a microcentrifuge at maximum speed for 1 minute. Cell lysates were then loaded at a concentration of 20 mg per variable into a gel. The gel was then run for 1.5 hours at 100 V and then transferred onto a methanol activated membrane. Once transferred, the membrane was blotted for the appropriate primary antibody in 5% milk and placed on a rocker in −80°C overnight. Following this exposure, the membranes were exposed to the appropriate secondary antibody for 3 hours in 5% milk at room temperature. After this exposure, the membranes were washed three times in TBST in increments of 10 minutes per wash. The membranes were then exposed to film for a total of 1 min. The film was then developed and labeled to mark results.Results Transient Knockdown Mia Paca‐2 cells were cultured in 6‐well plates at a confluency of 9.0×10^5 cells/well. Cells were then transfected transiently with 40 nM scramble siRNA or 40 nM HO‐1 siRNA. Cells were then incubated overnight at standard conditions and then harvested and lysed. Cell lysates were then collected and probed with SDS loading buffer and loaded into a gel at a concentration of 20 mg. After running and transferring the gel, the membrane was probed with anti‐HO‐1 or anti‐Actin antibody. Membranes were then developed on blue film showing a substantial knockdown in the levels of HO‐1 with steady, consistent bands of actin. Drug Administration Mia Paca‐2 cells were seeded in a 12‐well plate in 1.5 mL of media. The cells were incubated overnight at standard conditions. Cells were untransfected or transfected with 40 nM scramble siRNA or 40 nM HO‐1 siRNA. After incubation, stocks of various concentrations of Oxaliplatin were prepared. Each well was then treated with either DMSO or 1 mM of Oxaliplatin. Cell viability was then measured using Alamar Blue reduction for 2 hours at standard incubation conditions. Cell viability revealed a correlation in amount of cells killed when transfected for HO‐1 and treatment with Oxaliplatin. Lentiviral Transfection FTH‐1 expression in Mia Paca‐2 cells was stably reduced by lentiviral infection. Cells were cultured with the medium from infected 293T cells containing lentivirus. Cells with stably reduced levels of FTH‐1 were acquired by culturing in the presence of 5 mg/mL puromycin. Expression of FTH‐1 was then determined by Western Blot Anaylsis procedure. Cells lentivirally infected showed a decrease in FTH‐1 expression compared to those that were not lentivirally infected. Zinc Protoporphyrin‐IX Administration Mia Paca‐2 cells were cultured in 6‐well plates at a density of 9.0×10^5 cells/well and then incubated at standard conditions for 24 hours. After incubation, cells were subjected to either 0.0, 0.5, 1.0 or 2.0 mM of Zinc Protoporhyrin‐IX for 24 hours. Following, the cells were lysed and the lysate was collected and probed with SDS loading buffer. Cell lysate was then loaded into a gel at a concentration of 20 mg. Following the transfer of the gel to a membrane, the membrane was probed with FTH‐1, HO‐1, actin antibodies. Based on the results of the western blot analysis, it is evident that Zinc Protoporphyrin‐IX inhibits the expression of FTH‐1 while overexpressing the levels of HO‐1 in the Mia Paca‐2 cell line.Conclusions Decreasing HO‐1 expression in pancreatic tumor cells sensitizes pancreatic cancer cells to oxaliplatin. Reducing the expression of FTH‐1 transiently and stably enhances cytotoxic activities of oxaliplatin in pancreatic tumor cells. The HO‐1 enzymatic activity inhibitor zinc protoporphyrin‐9 promotes therapeutic effects of oxaliplatin. Support or Funding Information Funding for this project was provided by R25‐CA134283 grant from National Cancer Institute. This research was also supported by a Undergraduate Research Grant from Executive Vice President For Research and Innovation Internal Grant Program at University of Louisville.