Potential mechanisms of ascorbate-induced cytotoxicity in pancreatic cancer cells

1. Potential mechanisms of ascorbate-induced cytotoxicity in pancreatic cancer cells Juan Du, Garry R. Buettner, Larry W. Oberley, Joseph J. Cullen From the Departments of Surgery, Radiation Oncology and Free Radical & Radiation Biology Program, University of Iowa College of Medicine and VAMC, Iowa City, IA.

2. Abstract: Pharmacological concentrations of ascorbate easily achieved in humans may be effective in cancer therapeutics (PNAS, 104:8749, 2007). We hypothesized that ascorbate concentrations achievable with intravenous dosing may be cytotoxic in pancreatic cancer where the five year survival is < 3%. Pancreatic cancer cell lines were treated with ascorbate (0, 5, and 10 mM) for one hour and viability and clonogenic survival were determined. In addition, the immortal H6c7 cell line (pancreatic ductal epithelial cell) and its derivatives, H6c7eR-pBp (retroviral vector control), H6c7er-Kras (H6c7 cells expressing K-ras oncogene), and H6c7eR-KrasT (tumorigenic H6c7 cells expressing K-ras oncogene) (Cancer Res., 65:5045, 2005) were treated with ascorbate (0, 5, and 10 mM) and viability were determined. MIA PaCa-2 pancreatic cancer cell lines with functional mitochondria (rho+) and the same lines without functional mitochondria (rhoo) (J. Biol. Chem. 281:37416, 2006), were treated with ascorbate and clonogenic survival determined. The oxygen electrode method was used to determine H2O2 production. There was a time and dose-dependent increase in measured H2O2 production with increased concentrations of ascorbate. Ascorbate decreased clonogenic survival and viability in pancreatic cancer cell lines in a dose-dependent manner. Ascorbate had no effect on the H6c7 cell line, but decreased viability in the H6c7 cell lines that express K-ras oncogene. Ascorbate (5 and 10 mM) decreased viability in all human pancreatic cancer cell lines tested. In rho+ cells, ascorbate resulted in a dose-dependent decrease in clonogenic survival, but no cytotoxicity in the rhoo cells. We conclude that pharmacological doses of ascorbate, achievable in humans when given intravenously, may have potential for therapy in pancreatic cancer. The ascorbate-induced cytotoxicity in pancreatic cancer cells may be mediated by a mitochondrial mechanism. Support: NIH grants CA115785, CA66081, the Medical Research Service, Department of Veterans Affairs, and the Susan L. Bader Foundation of Hope.

3. Introduction • Pharmacological concentrations of ascorbate easily achieved in humans may be effective in cancer therapeutics (PNAS, 104:8749, 2007).

4. Predicted plasma Vitamin C concentrations in healthy persons after oral (top) or intravenous (bottom) administration of Vitamin C. Annals of Internal Medicine 140:533, 2004

5. Ascorbate as an anti-tumor agent. Ascorbate (vitamin C, ascorbate) is one of the early unorthodox therapies for cancer without supporting data. Initial published case reports demonstrated potential benefit from high dose ascorbate treatment. Subsequent reports documented the results of 100 patients with terminal cancer, in whom conventional therapy was no longer considered useful and were given intravenous ascorbate. Patients who received ascorbate survived 300 days longer than controls. A prospective study was then conducted randomizing patients to ascorbate treatment or palliative therapy. Treated patients had a median survival of 343 days vs. 180 days for controls. Smaller studies have also reported benefits of ascorbate. • To test whether ascorbate was effective, Moertel conducted two randomized placebo controlled studies randomized to oral ascorbate and neither study showed any benefit. Because Moertel’s studies were taken as definitive, ascorbate treatment was considered useless. However Moertel’s results were not comparable to those previous studies because ascorbate was given orally and not intravenously. • Emerging knowledge suggests that the role of ascorbate in cancer treatment should be reexamined. The evidence falls into two categories: clinical data on dose concentration relationships and laboratory data describing potential cell toxicity with high concentrations of ascorbate in vitro. Clinical data show that when ascorbate is given orally, fasting plasma concentrations are tightly controlled at < 100 M. As doses exceed 200 mg, absorption decreases, urine excretion increases and ascorbate bioavailability is reduced. In contrast, when 1.25 grams of ascorbate are administered intravenously, concentrations as high as 1 mM are achieved. Some clinicians have infused more than 10 grams of ascorbate in cancer patients and achieved plasma concentration of 1 to 5 mM. Thus, it is clear that intravenous administration of ascorbate can yield very high plasma levels, while oral treatment does not. • Chen et al. measured cell death in 10 cancer and 4 normal cell types using 1 hour exposures to ascorbate. Normal cells were unaffected by 20 mM ascorbate whereas 5 cancer cell lines had EC50 values of < 4 mM, a concentration achievable by intravenous administration. In addition, cell death was independent of metal chelators and dependent on H2O2 formation. H2O2 generation was dependent on ascorbate concentration, incubation time, and displayed a linear relationship with ascorbate radical formation. In vivo, Chen and colleagues demonstrated that intravenous injection of ascorbate (0.25-0.5 mg/g body weight) increased baseline concentrations of ascorbate in blood and extracellular fluid to > 8 mM and increased formation H2O2. These studies provides a foundation for pursuing pharmacologic ascorbate as a prooxidant agent in cancer therapy. J. Am. Coll. Nutrition 19:423, 2000

6. Pancreatic cancer therapy. Pancreatic cancer is the 4th most common cause of cancer death in the United States with over 33,000 fatal cases annually in the United States alone. Surgical resection of the primary tumor remains the only potentially curative treatment for pancreatic cancer. However, in population-based studies the number of patients undergoing resection with curative intent can be less than 3%. Even after resection, median survival is only 12-18 months and less than 20% of resected patients survive 5 years. The majority of patients die of metastatic cancer recurrence. • Other adjuvant treatments such as radiation therapy and chemotherapy, have not improved long-term survival after resection. The rate of chemotherapeutic response is less than 20%, while less than 10% of patients benefit from radiation therapy. • Because of the lack of poor therapeutic responsiveness of pancreatic cancer to surgery, chemotherapy, and radiation therapy, survival beyond five years is rare with median survival less than six months. • Thus, novel and effective therapies directed against pancreatic cancer are needed to control progression and metastatic disease. CA Cancer J Clin 57:43-66, 2007. Cur Probl. Surg 36:59-152, 1999.

7. Hypothesis • Ascorbate concentrations achievable with intravenous dosing may be cytotoxic in pancreatic cancer where the five year survival is < 3%.

8. Methods • Pancreatic cancer cell lines were treated with ascorbate (0, 5, and 10 mM) for one hour and viability and clonogenic survival were determined. • The immortal H6c7 cell line (pancreatic ductal epithelial cell) and its derivative H6c7er-Kras (H6c7 cells expressing K-ras oncogene) (Cancer Res., 65:5045, 2005) were treated with ascorbate (0, 5, and 10 mM) and viability were determined. • MIA PaCa-2 pancreatic cancer cell lines with functional mitochondria (rho+) and the same lines without functional mitochondria (rhoo) (J. Biol. Chem. 281:37416, 2006), were treated with ascorbate and clonogenic survival determined. • The oxygen electrode method was used to determine H2O2 production.

9. Mia PaCa-2 *P<0.05 vs 0 mM Ascorbate Arrow indicates that no colonies were formed when 20 mM ascorbate was given for one hour Clonogenic Survival (Clonogenic survival relative to 0 mM Ascorbate) * * Ascorbate

10. AsPC-1 *P<0.05 vs 0 mM Ascorbate * Clonogenic Survival (Clonogenic survival relative to 0 mM Ascorbate) * * Ascorbate

11. Figure 1. MIA PaCa-2 and AsPC-1 pancreatic cancer cells were treated with ascorbate (0-20 mM) for one hour and clonogenic survival determined. Ascorbate caused a dose-dependent decrease in clonogenic survival in pancreatic cancer cell lines.

12. A. C. D. B.

13. Figure 2. MIA PaCa-2 rho (o) cells depleted of mitochondrial DNA (mtDNA) were generated by incubating wild type cells (rho +) for 6-8 weeks with 100 ng/ml ethidium bromide. The medium was supplemented with 50 g/ml uridine and 100 g/ml pyruvate to compensate for the respiratory metabolism deficit. After selection, the MIA PaCa-2 rho (o) cells were cultured in the same medium without ethidium bromide. A. To verify mtDNA depletion, total cellular DNA was extracted and subjected to PCR using two pairs of human mtDNA specific primers: 1) Mts1 (forward) (5-cctagggataacagcgcaat-3) and Mtas 1 (reverse) (5 -tagaagagcgatggtgagag-3), which gave a 630-bp product, and 2) Mts2 (forward) (5-aacatacccatggccaacct-3) and Mtas2 (reverse) (5-ggcaggagtaatcagaggtg-3), which gave a 532-bp product. For control, we measured the expression of GAPDH, which is coded by nuclear DNA. B. Immunoblot of cytochrome c demonstrating that this protein which is coded by mtDNA is present in rho (+) but not in rho (o) cells. C. Immunoblots for manganese superoxide dismutase (MnSOD), copper/zinc SOD (CuZnSOD), thioredoxin (Trx), glutathione peroxidase (GPx1), and phospholipid glutathione peroxidase (PhGPx). D. Cells were incubated with DMSO and DMSO containing Antimycin A (AntA) 10 M for 15 minutes. Cells were stained for hydroethidine (DHE) and fluorescence measured by flow cytometry. Mean fluorescence intensity (MFI) calculated relative to rho (o) cells. Means  SEM, N = 3.

14. Mia PaCa-2 rho+ cells *P<0.05 vs 0 mM Ascorbate * Clonogenic Survival (Clonogenic survival relative to 0 mM Ascorbate) * * Ascorbate

15. Mia PaCa-2 rhoo cells *P<0.05 vs 0 mM Ascorbate Clonogenic Survival (Clonogenic survival relative to 0 mM Ascorbate) Ascorbate

16. Figure 3. Ascorbate (0-5 mM) demonstrated significant decreases in clonogenic survival in MIA PaCa-2 rho+ cells but no changes in clonogenic survival in MIA PaCa-2 rhoo cells.

17. * Surviving fraction (relative to no ascorbate for each treatment point)

18. *P<0.05 vs ascorbate * Surviving fraction (relative to control for each treatment point) * *

19. *P<0.05 vs ascorbate * Surviving fraction (relative to control for each treatment point) * * * *

20. Surviving fraction (relative to no ascorbate for each treatment point) *

21. * * Surviving fraction (relative to no ascorbate for each treatment point)

22. Surviving fraction (relative to no ascorbate for each treatment point) *

23. Figure 4. The addition of the mitochondrial electron transport chain (ETC) blocker Antimycin A (AntA) to ascorbate decreased human pancreatic cancer (MIA PaCa-2) clonogenic survival, relative to the use of the AntA alone. MIA PaCa-2 cells were treated for one hour with and without ascorbate 1 mM for one hour in the presence of Antimycin A 10 M for 4 hours. MIA PaCa-2 cells were treated with ascorbate are represented as clear bars per treatment group. Dark bars represent the surviving fraction of cells treated without ascorbate. P < 0.05 vs. no ascorbate, N=3.

24. *P < 0.05 vs 0 mM Ascorbate * * H2O2 (M) * Ascorbate

25. *P < 0.05 vs 0 mM Ascorbate * * * H2O2 (M) Time (min.)

26. Figure 5. H2O2 generation in cell culture medium. H2O2 was measured by oxygen electrode. H2O2 increased as a function of ascorbate concentration and a function of time (ascorbate 1 mM).

27. Viability (normalized to 0 mM for each cell line) * * * * * * * * * * *P<0.05 vs 0 mM for each cell line

28. Figure 6. Effects of pharmacologic ascorbic acid concentrations on pancreatic cancer and pancreatic ductal epithelial cells. All cells were treated with ascorbate (0, 5, 10 mM) for one hour. Cell viability determined by MTT assay. MIA PaCa-2, AsPC-1, BxPC-3 are pancreatic cancer cell lines. Immortalized pancreatic ductal epithelial cell line, H6c7 and its derivatives, and H6c7er-Kras (H6c7 cells expressing K-ras oncogene), also received ascorbate (0, 5, 10 mM) for one hour.

29. Conclusions • Pharmacological doses of ascorbate, achievable in humans when given intravenously, may have potential for therapy in pancreatic cancer. • The ascorbate-induced cytotoxicity in pancreatic cancer cells may be mediated by a mitochondrial mechanism.

30. Animal Protocol Day 1 Ascorbate 4 g/kg or Saline (1M) q day x 14 days 2 x 106 MIA PaCa-2 Pancreatic cancer cells Day 14 Stop treatment

31. Ascorbate (4 g/kg I.P.every day for 14 days). Hypertonic saline (g/kg I.P. every day for 14 days). Tumor volume (mm3) Days

32. Survival Days

33. Survival Days

34. *P< 0.01 vs control Surviving fraction (relative to control) *

35. *P< 0.01 vs control * * Surviving fraction (relative to control) *