Throughout most of the twentieth century, gastric cancer has been one of the leading causes of cancer death worldwide.^1,2 The main treatment is surgical resection, but most of advanced gastric cancer patients require systemic chemotherapy after surgery for adjuvant or palliative purposes. As systemic chemotherapy, cisplatin (cis-diaminedichloroplatinum, CDDP) is a widely used. In general, CDDP binds to DNA efficiently to form a variety of monoadducts and cross links either between adjacent bases on the same strand or opposing strands of DNA.^3,4 These DNA lesions contribute to the cytotoxicity of CDDP through blocking DNA replication and stimulating signals for apoptosis.⁵ However, the resistance to CDDP is developing while on chemotherapy, which is the major reason for the failure in chemotherapy. CDDP resistance can be the final phenomenon from multifactorial molecular mechanisms, which is not fully elucidated. One mechanism of resistance may be mediated through enhanced anti-apoptotic activity by activating Akt activity.⁶ In practice, to overcome initial resistance to CDDP, we usually add another cytotoxic chemotherapy that has different mechanisms of action and resistance.

As advances in medical science, our lifespan has been extended. The population of elderly cancer patients and their cancer-related mortality are expected to increase because the elderly have a high risk for cancer development.¹ However, these elderly patients cannot tolerate the conventional multi-combination chemotherapy. In turn, the most of elderly patients have experienced serious side effects from this combination chemotherapy. Some of these patients die of complications of chemotherapy. In this context, we need to find out less toxic agents in controlling cancer and enhancing the anti-cancer effects of the single anti-cancer agent.

Recently dietary agents are known to safely modulate physiological function and enhance anti-cancer activity.⁷?⁹ Apoptosis, is one of the anti-cancer mechanism of phytochemicals from fruits and vegetables, and is an active-energy requiring process (a type I programmed cell death) with distinctive phenotype such as blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation.^10,11 Its serve as defense mechanism for cancer development by eliminating damaged cells which are prone to develop cancer. Vitis coignetiae Pulliat (Meoru in Korea) traditionally has been used in Korean folk medicine for the treatment of inflammatory lesions and cancer. It contains abundance of anthocyanins belonging to a class of flavonoids. Recently, in vitro and in vivo anti-cancer activities of anthocyanins have been demonstrated regarding anti-angiognesis and cancer invasion.^12,13 We previously suggested that the anthocyanins isolated from Vitis coignetiae Pulliat (anthocyanidins isolated from meoru, AIMs) may induce apoptosis by suppressing Akt activity.¹⁴ Here, we determine whether AIMs can enhance anti-cancer effect of CDDP and investigate its involved mechanisms.

1. Cell culture and chemicals

Two of the gastric cancer cell lines, SNU-1, SNU-16 cells were obtained from the Laboratory of Cell Biology at the Cancer Research Institute in Seoul National University College of Medicine. They were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (Gibco BRL, Carlsbad, CA, USA), 100 U of penicillin and 100 μg/mL of streptomycin at 37°C in the humidified atmosphere of 95% air and 5% CO₂ in an incubator. Molecular mass markers for proteins were obtained from Pharmacia Biotech (Saclay, France). Antibodies against phospho-Akt (Ser473), Akt 1/2/3, X-linked inhibitor of apoptosis protein (XIAP), and procaspase 3 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Antibodies against phospho-Akt (Thr 308), and phospho-p53 (Ser 15) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Antibody against poly (adenosine diphosphate-ribose) polymerase (PARP) was purchased from BD Biosciences Pharmingen (San Diego, CA, USA). Antibody against β-actin was from Sigma (Beverly, MA, USA). Peroxidase-labeled donkey anti-rabbit and sheep anti-mouse immunoglobulin, and an enhanced chemiluminescence (ECL) kit were purchased from Amersham (Arlington Heights, IL, USA). All other chemicals not specifically cited here were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

2. Preparation of anthocyanins

Fruits of Vitis coignetiae Pulliat were collected at Jiri Mountain, Republic of Korea. Anthocyanins were isolated and characterized as previously described.¹⁵ The composition of AIMs was as follows: delphinidin-3, 5-diglucoside : cyanidin-3, 5-diglucoside : petunidin-3, 5-diglucoside : delphinidin-3-glucoside : malvdin-3, 5-diglucoside : peonidin-3, 5-diglucoside : cyanidin-3-glucoside : petunidin-3-glucoside : peonidin-3-glucoside : malvidin-3-glucoside = 3.5 : 3.4 : 7.1 : 23.9 : 8.0 : 9.6 : 9.1 : 16.1 : 5.7 : 13.4.

3. Cell proliferation assays

For the cell viability assay, the cells were seeded onto 24-well plates at a concentration of 5 × 10⁴ cells/mL, grown to 70% confluence and then treated with the indicated concentration of AIMs for 48 hours. Control cells were supplemented with media containing 0.1% dimethyl sulfoxide (vehicle control). Following treatment, cell viability was determined by MTT assays.

4. Western blotting

The procedure was performed as described previously.¹⁶ In brief, the concentrations of cell lysate proteins were determined by means of the Bradford protein assay (Biorad Lab, Ricmond, CA, USA) using bovine serum albumin as the standard. The protein (30 μg) was resolved by electrophoresis, electrotransferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA), and then incubated with primary antibodies followed by secondary antibody conjugated to peroxidase. Blots were developed with an ECL detection system.

5. Statistics

Each experiment was performed in triplicate. The results were expressed as means ± SD. Significant differences were determined using the one-way analysis of variance with post-test Newman-Keuls for the cases at least three treatment groups and Student’s t-test for two group comparison. Statistical significance was defined as P < 0.05. Statistical analysis was performed using IBM SPSS version 21.0 software (IMB Co., Armonk, NY, USA).

Anthocyanins isolated from Vitis coignetiae Pulliat (AIMs) suppress the proliferation of SNU-1 and SNU-16 cells and enhanced the anticancer effects cis-diaminedichloroplatinum

First, we assessed the effects of AIMs on the cell growth. MTT assay revealed that AIMs suppressed the proliferation of both the SNU-1 and SNU-16 cells in a dose-dependent manner (Fig. 1A and 1B). The degrees to inhibitory effects on the cell proliferation are similar in both cell lines. We previously found that SNU-1 cells are more sensitive to CDDP than SNU-16 cells at the high concentration that can induce apoptosis. At 100 μg/mL of AIMs, the difference in sensitivity to AIMs between SNU-1 and SNU-16 cells was not so much (Fig. 1C and 1D). Next, we checked the effects of CDDP and AIMs in the higher dose. As we previously showed higher CDDP (12 μg/mL) did not increased cell death in SNU-16 cells, but the increase in dose to 500 μg/mL of AIMs enhanced anticancer effects on SNU-16 cells, and also enhanced cytotoxicity of CDDP, but the enhanced cytotoxicity did not meet synergism (Fig. 1E and 1F) These results suggest that AIMs have anti-cancer cancer effects on SNU-16 cells which is resistant to CDDP.

cis-Diaminedichloroplatinum increased Akt activity, which was suppressed by anthocyanins isolated from Vitis coignetiae Pulliat

In this study, we first tested whether CDDP increases Akt activities. As shown in Figure 2A CDDP begin to increase Akt activities 2 hours after CDDP treatment. Next, we first tested whether AIMs inhibits the increased Akt activities. Western blot analysis revealed that AIMs inhibited the increased Akt activities in a dose-dependent manner (Fig. 2B). This study indicated that the inhibitory effects of AIMs on Akt may influence the anticancer effects of CDDP.

Anthocyanins isolated from Vitis coignetiae Pulliat enhanced cis-diaminedichloroplatinum-induced apoptosis by suppression of phosphorylation of Akt

Previously we reported that AIMs can induce apoptosis by reducing Bcl-2, XIAP through the inhibition of Akt-phosphorylation.¹⁴ To determine that AIMs enhance CDDP-induced apoptosis, we performed cell viability assay. As shown in Figure 3A, AIMs enhanced cytotoxicity of CDDP. To confirm this finding at the molecular level, we performed western blot analysis for apoptosis related factors and p-Akt. The suppression of Akt phosphorylation led to inhibition of XIAP and activation of apoptosis related enzyme (PARP and caspase 3). We found that AIMs enhance the cytotoxicity of CDDP by suppressing XIAP and Akt. This result suggests that AIMs can enhance the sensitivity of CDDP in p-53 mutant SNU-16 cells which is resistant to CDDP.

Recently, phytochemicals or food substances have been known to safely modulate and enhance anti-cancer activity.^8,17,18 We previously reported that AIMs can enhance apoptosis by suppression of Akt and anti-apoptotic proteins such as Bcl-2, and XIAP. In addition, Akt activation is accepted as one of major determinants of CDDP resistance in cancer cells. Moreover, Akt plays a critical role in regulating cell proliferation and apoptosis. Evidence suggest that Akt is also an upstream signal controlling Bcl-2 and IAP family members such as pro-apoptotic Bad and anti-apoptotic XIAP proteins, and its activity was regulated by phosphorylation.¹⁹ Therefore, it is reasonable to investigate whether AIMs can enhance the CDDP sensitivity in the SNU-16 which is resistant to CDDP. This study suggested that an increase in Akt activity is a cause of the failure of CCDP-induced apoptosis. Here, we need to check whether AIMs can suppress the increased Akt activity induced by CDDP. Consistent with previous studies showing that AIMs induce apoptosis by inhibiting Akt-phosphorylation,¹⁴ here AIMs suppressed the Akt-phosphorylation induced by CDDP. In addition to anti-Akt activity, AIMs has anti-epidermal growth factor receptor (EGFR) effects.²⁰ The anti-EGFR effects also contribute to suppress XIAP, an anti-apoptotic protein that we have shown in Figure 3B. We also tested another p-53 mutant MKN-28 human gastric cancer cells. The anthocyanins also enhance CDDP sensitivity of MKN-28 cells which is aggressive phenotypes and resistant to CDDP (data not shown). This is supporting our data that AIMs can enhance CDDP activity that is resistant to CDDP. In the present study, we have showed that AIMs enhances the CDDP-induced apoptosis in in p53 mutant SNU-16 cells (Fig. 4). In detail, AIMS enhanced the CDDP-induced apoptosis by induction of Bax as well as suppressing XIAP through the inhibition of Akt in SNU-16 cells. However, AIMs could not enhance sensitivity in p-53 wild SNU-1 cells. It is still under evaluation why the AIMs did not enhance anti-cancer effects of CDDP in SNU-1 cells.

The limitation is that there may be many other ways to enhance CDDP sensitivity using phytochemicals. Here, we are only focusing on Akt activities. Actually we previously found that p53 restoration can significantly enhance CDDP’s cytotoxic activities.²¹ Therefore, further investigation is required to find another way to enhance CDDP activities. Another limitation is that the maximum concentration used in this study appears to be too high to perform in vivo study. Actually we have shown the anti-cancer effects in vivo with AIMs.²² In addition, the maximal dose of the anthocyanins used in this study is consistent with those in many other studies on the anti-tumor effect of anthocyanins in culture cells.²³?²⁵

In conclusion, our results suggest that AIMs can enhance cisplatin induced apoptosis by suppression of Akt and XIAP in CDDP-resistant SNU-16 human gastric cancer cells.