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. Author manuscript; available in PMC: 2021 May 15.
Published in final edited form as: Int J Cancer. 2019 Oct 8;146(10):2822–2828. doi: 10.1002/ijc.32658

A Chirality-Dependent Action of Vitamin C in Suppressing KRAS Mutant Tumor Growth by the Oxidative Combination: Rationale for Cancer Therapeutics

Xinggang Wu 1, Mikyung Park 1, Dilara A Sarbassova 1, Haoqiang Ying 1,2, Min Gyu Lee 1,2, Rajat Bhattacharya 3, Lee Ellis 2,3, Christine B Peterson 4, Mien-Chie Hung 1,2, Hui-Kuan Lin 5, Rakhmetkazhi I Bersimbaev 6, Min Sup Song 1,2, Dos D Sarbassov 1,2,7
PMCID: PMC7365556  NIHMSID: NIHMS1557421  PMID: 31472018

Abstract

Kirsten rat sarcoma (KRAS) mutant cancers, which constitute the vast majority of pancreatic tumors, are characterized by their resistance to established therapies and high mortality rates. Here, we developed a novel and extremely effective combinational therapeutic approach to target KRAS mutant tumors through the generation of a cytotoxic oxidative stress. At high concentrations, Vitamin C (VC) is known to provoke oxidative stress and selectively kill KRAS mutant cancer cells, although its effects are limited when it is given as monotherapy. We found that the combination of VC and the oxidizing drug arsenic trioxide (ATO) is an effective therapeutic treatment modality. Remarkably, its efficiency is dependent on chirality of VC as its enantiomer D-VC is significantly more potent than the natural L-VC. Thus, our results demonstrate that the oxidizing combination of arsenic trioxide and D-VC is a promising approach for the treatment of KRAS mutant human cancers.

Keywords: Kirsten rat sarcoma (KRAS) mutant cancer cells, reactive oxygen species (ROS), oxidative stress, drug combination, apoptosis

Introduction

Kirsten rat sarcoma (KRAS) mutant cancers represent highly malignant oncologic disorders with poor clinical outcomes. Activating KRAS mutations occur predominately in pancreatic cancer and are found in almost all (95%) of pancreatic ductal adenocarcinomas (PDACs) 1. They are also common in biliary tract (33%), colorectal (32%), lung (19%) and ovarian (17%) cancers 2. However, no effective therapies have been developed to treat KRAS mutant cancers, because the KRAS gene encodes a small GTPase with no distinctive pocket for targeting 3, 4. Hence, a specific and potent means of targeting this highly malignant oncogenic pathway is one of the most challenging and demanding tasks in oncology.

KRAS mutations lead to hyperactivation of the mitogen-activated protein kinase and phosphatidylinositol-3-OH kinase pathways, which accelerates cancer cell growth and proliferation. KRAS-dependent transformation results in a distinctive cellular metabolism with an unbalanced redox state due to high glucose consumption 5, 6 leading to elevated generation of reactive oxygen species (ROS) 7. A vulnerable redox state has been actively explored as a therapeutic avenue to target KRAS mutant cancer cells by inducing oxidative stress 8, 9. Vitamin C ([VC] or also known as ascorbic acid) has attracted attention for its anticancer activity since 1974 10, 11 and a recent study revealed its selective action to induce oxidative stress at high concentrations in KRAS mutant cancer cells 9. A selective effect of VC on the cancer cells was linked to an elevated glucose uptake mediated by abundant expression of the glucose transporter GLUT1 12, 13. In the body, VC is oxidized to dehydroascorbate (DHA), which is actively absorbed by the cancer cells through GLUT1 because of its structural resemblance to glucose. Inside the cell, DHA is reduced back to VC at the expense of oxidizing glutathione (GSH). As a result, an extensive DHA reduction in KRAS mutant cells leads to oxidative stress 9.

Materials and Methods

Cell culture and cell lines

The human HCT116 (Research Resource Identifier [RRID]:CVCL_0291), RKO (RRID:CVCL_0504), HT-29 (RRID:CVCL_0320), HPAC (RRID:CVCL_3517) and MRC-5 (RRID:CVCL_0440) cell lines were obtained from the American Type Cell Culture Collection (ATCC, Rockville, Maryland, USA). Mouse AK192 and human HKh2 (RRID:CVCL_9797) cells were derived from HCT116 cell line by deleting the KRAS mutant allele were described previously and provided from the original sources by Drs. Haoqiang Ying and Senji Sirasawa 6, 14. All studied human cell lines have been authenticated using the short tandem repeat profiling within the last three years and all culture experiments were performed with mycoplasma-free cells. Cells were cultured at 37°C in a humidified incubator and maintained in the DMEM/Ham’s F12 medium (catalog #DFL13 from Caisson Labs) containing 17.5 mM of glucose and supplemented with 10% FCS, 2 mM glutamine and penicillin (100 units/ml)-streptomycin (100 μg/ml). For the drug treatments, the cells were split to 50% confluency previous day in the same medium containing 17.5 mM glucose supplemented with 10% FCS, 2 mM glutamine and penicillin (100 units/ml)-streptomycin (100 μg/ml); the cells were then incubated with the indicated concentrations of the drugs. For HCT116 cells to obtain 50% confluency, 500,000 (a half million) cells were plated into six-well cell culture dishes in 2-ml cell culture medium a day prior the drug treatment.

Drug preparations

The following drugs were obtained from Sigma: Vitamin C ([VC] or Ascorbic Acid, catalog #A7506), D-Vitamin C (d-(−) isoascorbic acid, catalog #856061) and Arsenic Trioxide ([ATO], catalog #A1010). We prepared 340 mM stock of VC by dissolving 30 g of VC in 400 ml of the autoclaved phosphate-buffered saline (PBS), stirring and then slowly adding 39 g of sodium bicarbonate (NaHCO3, catalog #S5761) while continuously stirring for 5 min as described previously 15. The addition of sodium bicarbonate changed the acidic pH of VC from pH 2.3 to a physiological pH 7.35 that was critical for injections into mouse. After checking the pH, we adjusted the VC solution to 500 ml by PBS. The solution was further filtered, aliquoted, and stored in a −20oC freezer. Similar steps were taken to prepare 340 mM stock of D-VC. The stock of ATO was prepared at a 330 mM concentration by dissolving 13 g of ATO in 1 N sodium hydroxide (NaOH) and adjusting the total volume to 200 ml. After the ATO had been dissolved, it was filtered, aliquoted and stored in a −20oC freezer.

Apoptosis assay

Cell apoptosis was evaluated by flow cytometry. The cells were harvested 48 hours after treatment with VC, ATO or both. The cells were resuspended at a density of 1×106 cells/ml in 1x binding buffer after three PBS washes. The cells were then stained with FITC-Annexin V and propidium iodide (PI) for 30 minutes at 4oC in the dark using the FITC Annexin V: PE Apoptosis Detection Kit I (BD Biosciences). The cells were analyzed using BD FACSCanto II flow cytometer according to the manufacturer’s instructions to detect early and late apoptosis. All experiments were performed in triplicate.

Measurement of intracellular ROS

HCT116 cells were treated with VC, D-VC, ATO or combination of VC or D-VC with ATO for 72 hours and then collected by trypsinization and incubated with MitoSOX (2μM) for 30 minutes at 4oC in the dark before being analyzed by flow cytometry.

Animal study and Immunohistochemical analysis

The animal study complied with the protocol that has been approved by the University of Texas MD Anderson Cancer Center (#0000193). 1.5 million HCT116 cancer cells were transplanted into nude mice subcutaneously, and the drugs were injected after 10 days, when tumors became larger than 0.8 cm in diameter. The excised xenograft tumors and livers were examined by the immunohistochemical analysis. Formalin-fixed, paraffin embedded tissues were stained with Hematoxylin and Eosin. For immunohistochemical analysis, tumor tissue slides were incubated in a microwave oven in citrate buffer (pH 6.0) for 15 minutes for epitope retrieval. The slides were then incubated with cleaved caspase 3 (Cell Signaling Technology, product number 9661, 1:100 dilution) at 4°C for 18 hours. After the secondary antibody incubation, the slides were dehydrated and stabilized with mounting medium and the images were acquired with a Leica DM1000 microscope. Cleaved caspase 3-positive cells were counted at x400 magnification in five or six randomly selected areas in tumor samples.

Dual Drug Combination Assay

HCT116 cells were plated in 96-well plates and treated with various concentrations of VC, either alone or in combination with ATO, for 48 hours. Cell viability was determined using a colorimetric MTT assay. The cell culture medium was replaced prior to the application of MTT solution because the presence of VC in the medium interfered with the dye staining. Synergistic effects were determined using the Chou-Talalay method to calculate the CI 16, 17.

Data Availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

Statistical analysis

Statistical significance was determined using an unpaired Student’s t test or analysis of variance (ANOVA) by GraphPad Prism 6 (Graph Pad Software). Data were considered to be significant when P values are <0.05. Sample sizes and animal numbers were chosen on the basis of the results of pilot studies performed in the laboratory.

Results

The ATO and VC combination is cytotoxic to the KRAS mutant cancer cells

We assumed that if VC alone is sufficient to induce oxidative stress in KRAS mutant cancer cells, the combination of VC and another oxidizing reagent would effectively enhance oxidative stress and lead to cytotoxic effects with high selectivity towards the cancer cells. We selected a potent oxidizing drug arsenic trioxide (ATO) as the second compound, which is effective in treating acute promyelocytic leukemia (APL) 18. To test this hypothesis, we treated the human HCT116 (KRAS mutant) cancer cells were treated with 1 mM VC, 5 μM ATO or both. We observed substantial cell death only in cells treated with the drug combination, as visualized by a massive detachment of cells (Fig. 1A), suggesting that the ATO/VC combination had cytotoxic impact.

Figure 1.

Figure 1.

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Induction of apoptosis by the ATO and VC combination in the KRAS mutant cancer cells. a The images of HCT116 human cancer cells following the treatment with PBS (control), 1 mM VC, 1mM D-VC, 5 μM ATO, 1 mM VC+5 μM ATO or 1 mM D-VC and 5 μM ATO for 48 hours. b The cell death assay histograms of the cells described in (a) using Annexin-V and PI staining and flow cytometric analysis. c The graphical presentation of the dead cell analysis shown in (b).

An analysis of the detached cells using the Annexin V/propidium iodide (PI) flow cytometric apoptotic assay indicated that the cells were apoptotic 19. The cells treated with VC alone showed basal cytotoxicity of 12.5% as compared to 13% in the non-treated control cells (Figs. 1B and 1C). Previously, it was shown that the cytotoxic effect of VC in HCT116 cells was glucose dependent and that VC was highly effective when cells were maintained in a low glucose (2 mM) medium 9. In the cell culture medium containing a physiological glucose concentration (5.5 mM), VC alone did not induce a cytotoxic effect. Besides, ATO alone induced only a moderate increase in cytotoxicity (17.2%), but the combination of ATO and VC induced a potent apoptotic response with 75.4% of cells undergoing apoptosis after 48 hours of treatment (Fig. 1B and 1C). The combined action of ATO and VC resulted in a synergistic apoptotic impact as indicated by a substantial (45.7%) increase in cell death above their additive cytotoxic effect (29.7%). The synergistic action of ATO and VC was further supported by the results of our combinational index (CI) study, which revealed CI values less than 1 (CI<1), as calculated by the Chou-Talalay method 16, 17, shown in Supplementary Figure 1. Of note, the effect of VC was not restricted to its natural L-optical isomer (L-VC) since its enantiomer (D-VC) showed similar behavior inducing potent cytotoxicity (68.1%) in cells only in combination with ATO (Figs. 1B and 1C). These findings indicate that both optical isomers of VC (L-VC or D-VC) act synergistically with the oxidizing drug ATO to induce the effective elimination of HCT116 cancer cells that are known to carry the oncogenic KRASG13D allele 14.

Sensitivity to the oxidative combination is dependent on the oncogenic KRAS expression

To determine whether the observed pronounced sensitivity of HCT116 (KRASG13D mutant) cancer cells to the combination of ATO/VC is indeed linked to the oncogenic KRAS allele, we used an isogenic cell line HKh2, which is derived from HCT116 cells that have a deleted KRAS mutation 14. The combination of ATO and VC in the HKh2 cells induced a much weaker apoptotic response, without a potentiating effect (as was 4.7% below of the additive effect of drugs). The cytotoxic response in HKh2 cells (Supplementary Fig. 2) to the drug combination was six-fold lower than that observed in the parental (HCT116) KRAS mutant cells (Supplementary Fig. 3) even at a higher drug dosage. Conversely, AK192 cells (a mouse PDAC cell line) expressing oncogenic KrasG12D6 were highly sensitive to the ATO/VC combination, as observed by the induction of a potent cytotoxic impact in the range of 91% (Supplementary Fig. 4). A high sensitivity to the ATO/VC combination was also observed in HPAC cells, the human PDAC cell line carrying the KRASG12D mutant allele 20, with 56.7% of cells undergoing cell death (Supplementary Fig. 5). The drug combination targeted KRAS mutant cancer cells with high specificity, as we did not observe a potent cytotoxic impact of the drug combination in KRAS wild-type HT29 (17.4%) or RKO (22.2%) colorectal cancer cells (Supplementary Figs. 6 and 7) or human MRC5 primary fibroblasts (14.4%), as shown in Supplementary Figure 8. These results indicate that ATO and VC act synergistically to induce a potent cytotoxic effect selectively in KRAS mutant cancer cells.

If ATO and VC work together by inducing cytotoxic oxidative stress that will indicate an excessive generation of ROS will accompany the action of drugs in KRAS mutant cancer cells. To determine whether the generation of ROS is provoked by the drug combination, we transiently incubated HCT116 cancer cells with the superoxide-dependent fluorogenic dye MitoSOX Red following the drug treatments. The flow cytometry analysis revealed basal ROS accumulation in approximately 5% of the control and VC or D-VC treated cells. Up to 26% more ROS was detected in the ATO treated cells, whereas an increase of up to 94% was observed in cells treated with the ATO/VC or ATO/D-VC combination (Figs. 2A and B). Most of the cells treated by both drug combinations had high ROS levels and underwent active cell death (Fig. 1). It is likely that the accumulation of ROS in most cells treated with the drug combinations reflects not only the apoptotic (Annexin V and PI-positive) cells but also the necrotic (PI-positive ) or early apoptotic (Annexin V positive) cells that are known to be also associated with the generation of ROS 21–23. To link a cytotoxic impact of the oxidative drug combination to ROS generation, the cells were incubated with the drugs and reducing compound N-acetyl cysteine (NAC)24. We found that NAC was effective in blocking not only generation of ROS but also the cytotoxic impact caused by the drug combination (Supplementary Figure 9). Thus, a cytotoxic impact induced by the drug combination in KRAS mutant cancer cells is mediated by generation of ROS.

Figure 2.

Figure 2.

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Cytotoxic oxidative stress is induced by the ATO and VC combination in the KRAS mutant cancer cells. a ROS-detecting histograms of the cells with the similar treatment described in the Figure 1. HCT116 cancer cells were treated with PBS (control), 1 mM VC, 1mM D-VC, 5 μM ATO, 1 mM VC and 5 μM ATO or 1 mM D-VC and 5 μM ATO for 48 hrs and following treatments were incubated with 2μM MitoSOX for 30 minutes and analyzed by flow cytometry. b The graphical presentation of the ROS detection analysis by Mitosox is shown in (a).

A chirality dependent action of VC in suppressing KRAS mutant tumor growth

Consistent with this hypothesis, the action of ATO is significantly potentiated when it is combined with VC, which leads to cytotoxic oxidative stress selectively in KRAS mutant cancer cells. Considering that the ATO/VC combination is effective in cell culture, we tested it in a xenograft mouse cancer model. HCT116 cancer cells were transplanted into nude mice subcutaneously, and the drugs were injected after 10 days, when tumors became larger than 0.8 cm in diameter. The stocks of VC and D-VC for mouse injections were prepared by adding sodium bicarbonate according to the method used in a previous study 15.

To increase the efficacy of the drug combination, we introduced a 2-hour time interval between the ATO and VC injections. In addition, to eliminate the blood glucose fluctuations in mice during drug administration, food was withdrawn 2 hours prior to the ATO injections and replaced 2 hours after the VC injections. In this manner, the dose of VC was decreased from a reported daily dosage of 4 g/kg 9 to 1.5 g/kg, which was not toxic in combination with ATO.

Within the first week of injections, we observed massive tumor shrinkage in mice that had received the injections of the ATO/D-VC combination. After the ninth injection, all five mice in the combination group showed massive tumor shrinkage to the degree that macroscopic identification was only possible because of the development of scar tissue. Only two scarred tumors were observed in the group of mice injected with D-VC alone (Fig. 3A). However, mice injected with ATO or ATO and VC exhibited formation of initial scars on some tumors only after the twelfth injection (Supplementary Figure 10). After the 15th (final) drug injection, a tumor analysis indicated that the ATO/D-VC combination was the most effective treatment in suppressing tumor growth, with an average tumor weight at least 70% (3.4 fold) lower than that in the control group (Figs. 3B and C). Suppression of tumor growth was also detected in mice treated with the ATO/VC combination where: tumor weights were 44% (1.77 fold) lower than were those in the control group. In contrast, ATO alone resulted in an only 20% decrease in tumor weight and VC and D-VC were even less effective with high variations. Consistent with tumor size effects, the tumors from mice treated with the ATO/D-VC combination had the highest rate of apoptosis (as determined by cleaved caspase 3 detection) compared to those from mice treated with a single agent or the ATO/VC combination (Supplementary Figures 11 and 12). Importantly, we did not observe a substantial drug induced toxicity because the histological analysis of mouse livers did not detect any adverse abnormalities or signs of necrosis (Supplementary Figure 13) that was coherent with no lethality or weight loss in mice (Supplementary Figure 14). Thus, we found that, contrary to the results of the cell culture studies, a non-natural enantiomer of VC (D-VC), in combination with ATO, was far more potent in suppressing KRAS mutant tumor growth than was the natural VC form (L-VC).

Figure 3.

Figure 3.

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A stereodependent action of VC in the suppression of KRAS mutant tumor growth by the ATO and VC combination. a The images of xenograft tumors after nine drug injections. HCT116 human cancer cells were transplanted into nude mice. After 10 days when the tumors had reached at least 0.8 cm in diameter, the mice were injected daily with PBS (control), ATO (7 mg/kg), VC (1.5 g/kg), D-VC (1.5 g/kg), VC and ATO or D-VC and ATO. b Images of the excised xenograft tumors shown in (a) following the final (15th) drug injection. c Graphical presentation of the weight of the xenograft tumors shown in (b). One-way analysis of variance (ANOVA) confirmed a significant difference in tumor weight across the six groups (p = 1.02e-06***).

Discussion

We report here that ATO and VC in combination act synergistically by selectively inducing a cytotoxic oxidative stress in KRAS mutant cancer cells. The mechanism of a self-destructing ROS generation remains unknown, but it is becoming evident that KRAS mutant cancer cells accumulate ROS under oxidative stress. An elevated glucose consumption rate by these highly malignant cancer cells is likely to impose a metabolic state that is sensitive to oxidative stress. The indicated working concentration of VC at one millimolar concentration does not limit a potential clinical application of the ATO and VC combination because the intravenous VC administration bypassed limitation of its oral administration by increasing the drug dosage hundred times and by reaching maximum safe administration of VC to 10 millimolar 25. Importantly, we found a remarkable difference in efficacy between the L-VC and D-VC enantiomers when they were combined with ATO in the xenograft mouse model, while both combinations showed similar potency according to the cell culture study. This finding suggests that both VC isomers use a similar mechanism in KRAS mutant cancer cells via provoking an oxidative stress but the distinctive pharmacokinetics of D-VC made it more superior to its natural L-VC form in the animal model. It has been reported that the oxidizing of VC to DHA is a chirality-dependent reaction that occurs at an approximately eight times faster rate for L-VC 26. It is possible that a slow oxidation of D-VC results in a more sustained accumulation of DHA in the blood circulation and subsequently promotes a higher absorbance of DHA by the cancer cells. While a chirality specific pharmacokinetics of VC have yet to be defined, we demonstrated that ATO and D-VC represented a very potent combination for targeting KRAS mutant cancer cells in the mouse xenograft model.

In summary, we identified a promising oxidizing drug combination in the treatment of KRAS mutant human cancers or other cancers with high glucose consumption. The potential clinical benefits of the VC enantiomers in combination with ATO have yet to be explored.

Supplementary Material

Supp Fig S1-S14

Novelty & Impact Statements.

The Kirsten rat sarcoma (KRAS) mutations lead to highly malignant cancers. Development of novel treatment strategies by exploiting distinctive sensitizing features endowed on cancer cells by KRAS mutations is urgently needed because direct targeting approach remains unresolved. Our studies show that induction of a cytotoxic oxidative stress by the drug combination is a potent and promising therapeutic approach to target KRAS mutant cancers that revealed a chirality-dependent action of Vitamin C in tumor suppression.

Acknowledgements

We are grateful to Dr. Senji Sirasawa for providing the HKh2 cancer cell line for this study, to Dr. Ergun Sahin (Baylor College of Medicine, Houston, TX) for the helpful discussions and scientific editing the manuscript and to Ann Sutton from the Department of Scientific Publications of MDACC for editing the manuscript. This work was supported by funding provided by the Cancer Prevention & Research Institute of Texas (CPRIT) grants RP130276 and RP140408, Internal Research Grant of MDACC (D.D.S.) and the NIH R01 grants CA207098 and CA207109 (M.G.L.) and also the startup funding from Nazarbayev University.

Grant sponsor: Cancer Prevention & Research Institute of Texas (CPRIT), National Institute of Health (NIH) and startup funding of Nazarbayev University.

Abbreviations

KRAS

Kristen rat sarcoma

VC

Vitamin C

L-VC

L-optical isomer of VC

D-VC

D-optical isomer of VC

PDAC

pancreatic ductal adenocarcinomas

ROS

reactive oxygen species

DHA

dehydroascorbate

ATO

arsenic trioxide

CI

combinational index

Footnotes

Conflict of interest: The authors declare no conflict of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp Fig S1-S14
NIHMS1557421-supplement-Supp_Fig_S1-S14.pdf (6.9MB, pdf)

Data Availability Statement

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

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