Diallyl Trisulfide Inhibits Growth of NCI-H460 in Vitro and in Vivo, and Ameliorates Cisplatin-Induced Oxidative Injury in the Treatment of Lung Carcinoma in Xenograft Mice

Abstract

Diallyl trisulfide (DATS), an organosulfuric component of garlic oil, exhibits potential anticancer and chemopreventive effects. Cisplatin (DDP), a common chemotherapeutic agent, has provided great therapeutic contributions to treating solid tumors, but with serious side effects. Here, we verified the anti-tumor properties of DATS on lung cancer in vitro and in vivo, and evaluated synergistic effects of DATS combined with DDP on the NCI-H460 xenograft model. Significantly decreased cell viabilities, cell cycle G₁ arrest, and apoptosis induction were observed in DATS treated NCI-H460 cells (p<0.05). And injection of DATS (30 or 40 mg/kg) to female Balb/c mice significantly inhibited the growth of human NCI-H460 cell tumor xenograft (p<0.001). Moreover, DATS in combination with DDP exhibited enhanced anti-tumor activity via induction of apoptosis. Apoptosis pathways were confirmed by modulation of p53, Bcl-2 family members; induction of active caspase-3/8/9 and activation of JNK- and p38-MAPK pathways. Interestedly, DATS+DDP administration exerted fewer side effects, such as suppressing the weight loss and ameliorating DDP-induced oxidative injury, especially in renal parenchyma. In addition, increased E-cadherin and decreased MMP-9 expression levels were observed in DATS-treated tumor tissues. These studies provide supports that DATS might be a potential candidate for combination with DDP in cancer treatment.

Keywords: Diallyl trisulfide; attenuate side effect.; cisplatin; enhanced effect; lung carcinoma.

Figure 1

DATS inhibits cell growth and induces G0/G1 phase arrest in NCI-H460 cells. (A) Effect of DATS on cell viability. (B and C) Flow cytometry analysis of cell cycle distribution affected by DATS. Percentage of cells in each phase of cell cycle was estimated by Modfit software. A typical apoptosis sub-peak was observed after DATS treatment. (D) Western blot analysis of the level of the G0/G1-related proteins. Densitometric quantification was performed by AlphaView software. Data are presented as the mean ± SEM. **p<0.01, ***p<0.001 vs. control group.

Figure 2

DATS induced apoptosis in NCI-H460 cells. (A) Fluorescent micrographs of DATS-treated NCI-H460 cells after DAPI staining. Scale bar = 20 µm. (B and C) Flow cytometric analysis of apoptosis in NCI-H460 cells after 24 h of DATS treatment. The early and late apoptosis was quantified by CXP software. Data are presented as the mean ± SEM. **p<0.01, ***p<0.001, ****p<0.0001 vs. control group.

Figure 3

Western blot analysis of the levels of the apoptosis-related protein in DATS-treated NCI-H460 cells (A) Regulation of p53, Cytochrome C and Bcl-2 family proteins (B) Activation of caspases and the degradation of PARP. Data are presented as the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 vs. control group.

Figure 4

DATS inhibits tumor growth and enhances the efficacy of cisplatin (DDP) in xenograft tumors. Tumor-bearing mice (7 animals per group) were treated by intraperitoneal injection as follows: vehicle control, DATS groups (20, 30, 40 mg/kg/day), DDP (4 mg/kg/5 days) and a combination of DDP and DATS (20, 30 mg/kg) for 24 days. (A and B) The changes of tumor volume and body weight in DATS treatment and combination treatment groups. *p<0.05, **p<0.01, ***p<0.001.

Figure 5

DATS induced apoptosis in tumor xenografts via activation caspase pathway. (A) Representative micrographs of tumor sections: hematoxylin and eosin (H&E) staining, TUNEL staining and immunohistochemistry (IHC) for Ki-67, Bax, p21 and p53. Magnification × 400, Scale bar = 20 μm. (B) Effect of DATS on expression of MMP-9 and E-cadherin, *p<0.05, **p<0.01, ***p<0.001 vs control group. (C) Western blot analysis of apoptosis markers, such as PCNA, FADD, cleaved PARP and cleaved caspase-3. The induction fold of proteins was calculated as the intensity of the treatment relative to that of control by densitometry.

Figure 6

The toxicity of DATS and combined with DDP in treatment of mice bearing NCI-H460 tumor (A and B) The changes of major organ (spleen and kidney) indexes of mice at the end of treatment. *p<0.05, **p<0.01, ***p<0.001. (C) Histological analysis of kidney, original magnification, 400×, and scale bar is 20μm. Normal cortical structure is observed in the renal sections of both control and DATS-treated mice. Glomerular atrophy (G), wide capsular space (arrow), wide lumina (*) with accumulation of homogenous hyaline casts and interstitium inflammatory cell infiltration (I) are noticed in DDP-treated mice. Wide capsular lumina (arrow) and wide lumina (*) are seen in the combined DATS- and DDP-treated mice.

Figure 7

Modulation of intracellular signaling molecules involved in inflammatory and apoptosis in NCI-H460 tumor tissues. Effect of DATS on expression of phosphorylated and total Akt, ERK1/2, JNK and p38. The data are presented as the mean ± SEM (n=3), *p<0.05 and **p<0.01.