Mechanical stress promotes cisplatin-induced hepatocellular carcinoma cell death

Abstract

Cisplatin (CisPt) is a commonly used platinum-based chemotherapeutic agent. Its efficacy is limited due to drug resistance and multiple side effects, thereby warranting a new approach to improving the pharmacological effect of CisPt. A newly developed mathematical hypothesis suggested that mechanical loading, when coupled with a chemotherapeutic drug such as CisPt and immune cells, would boost tumor cell death. The current study investigated the aforementioned mathematical hypothesis by exposing human hepatocellular liver carcinoma (HepG2) cells to CisPt, peripheral blood mononuclear cells, and mechanical stress individually and in combination. HepG2 cells were also treated with a mixture of CisPt and carnosine with and without mechanical stress to examine one possible mechanism employed by mechanical stress to enhance CisPt effects. Carnosine is a dipeptide that reportedly sequesters platinum-based drugs away from their pharmacological target-site. Mechanical stress was achieved using an orbital shaker that produced 300 rpm with a horizontal circular motion. Our results demonstrated that mechanical stress promoted CisPt-induced death of HepG2 cells (~35% more cell death). Moreover, results showed that CisPt-induced death was compromised when CisPt was left to mix with carnosine 24 hours preceding treatment. Mechanical stress, however, ameliorated cell death (20% more cell death).

Figure 1

The effect of CisPt (0.5–32 μg/mL) on HepG2 cells. (a) Cell viability of HepG2 cells after exposure to CisPt (0.5–32 μg/mL) for 24, 48, and 72 hours as per MTT assay. The data are presented as a mean of at least three independent experiments. Error bars represent one standard error of the mean (P < 0.05). (b) Western blot analysis showing full-length and cleaved PARP-1 expression in HepG2 cells after exposure to different factors. UV = HepG2 cells exposed to ultraviolet light, C32 = cells exposed to CisPt 32 μg/mL, C16 = cells exposed to CisPt 16 μg/mL, and Control = HepG2 cells not exposed to any factor (negative control). (c) The percentage of full-length PARP-1 and cleaved PARP-1 as compared to the total PARP-1. The extent of PARP-1 cleavage was quantified by measuring relative intensities of the cleaved PARP-1 bands in the Western blot of Figure 1(b) and comparing them to intensity of the full-length PARP-1.

Figure 2

HepG2 cell survival rate after exposure to different factors for 24 hours exposure. Control = cells not exposed to any factor (negative control), C = CisPt 2 μg/mL, P = PBMCs, and M = mechanical stress. Error bars represent one standard error of the mean (P < 0.05).

Figure 3

RNA expression of apoptosis-related genes BAX, Caspase-3, XIAP, HSP70, and the internal control β-Actin after exposure of HepG2 cells to different factor(s). Columns from left to right are unstressed cells (negative control), HepG2 cells exposed to UV radiation (positive control), and HepG2 cells exposed to different factor(s). (a) HepG2 cells exposed only to CisPt 2 μg/mL for 24 hours. (b) HepG2 cells cocultured with PBMCs in 3 : 1 ratio (PBMCs : HepG2 cells) for 24 hours. (c) HepG2 cells cocultured with PBMCs in 3 : 1 ratio (PBMCs : HepG2 cells) and simultaneously exposed to CisPt 2 μg/mL for 24 hours. (d) HepG2 cells exposed only to mechanical stress at 300 rpm for 24 hours. (e) HepG2 cells simultaneously exposed to CisPt 2 μg/mL and mechanical stress at 300 rpm for 24 hours. (f) HepG2 cells cocultured with PBMCs in 3 : 1 ratio (PBMCs : HepG2) and simultaneously exposed to mechanical stress at 300 rpm for 24 hours. (g) HepG2 cells cocultured with PBMCs in 3 : 1 ratio (PBMCs : HepG2) and simultaneously exposed to CisPt 2 μg/mL plus mechanical stress at 300 rpm for 24 hours. (h) Caspase-3 RNA levels as indicated by the number of folds relative to the Control. Control = cells not exposed to any factor (negative control), C = CisPt, P = PBMCs, M = mechanical stress, and UV = cells exposed to ultraviolet (positive control). Error bars represent one standard error of the mean.

Figure 4

Morphological changes at 200x magnification for HepG2 cells exposed to different factors. Control = cells not exposed to any factor (negative control), C = CisPt, P = PBMCs, and M = mechanical stress.

Figure 5

Different carnosine : CisPt molar ratios cytotoxic effect on HepG2 cells for 24 hours as per MTT assay. The data are presented as a mean of at least three independent experiments (mean ± SD). Left to right: Media = control HepG2 cells. Car : Cis 1 : 1 = HepG2 cells exposed to CisPt 8 μg/mL and carnosine in 1 : 1 molar ratio. Car : Cis 1.5 : 1 = HepG2 cells exposed to CisPt 8 μg/mL and carnosine in 1.5 : 1 molar ratio. Car : Cis 2 : 1 = HepG2 cells exposed to CisPt 8 μg/mL and carnosine in 2 : 1 molar ratio. Car : Cis 2.5 : 1 = HepG2 cells exposed to CisPt 8 μg/mL and carnosine in 2.5 : 1 molar ratio. Car : Cis 3 : 1 = HepG2 cells exposed to CisPt 8 μg/mL and carnosine in 3 : 1 molar ratio (error bars represent the standard deviation). The P values shown are the values of the mean of every condition as compared to the control cells (^*** P < 0.001).

Figure 6

Cell viability of HepG2 cells after exposure to CisPt (8 μg/mL) and carnosine (1 : 3 molar ratio) with and without mechanical shaking (300 rpm) for 24 hours as per MTT assay. The data are presented as a mean of at least three independent experiments (mean ± SD). Left to right: Media = control HepG2 cells. C8 = HepG2 cells exposed to CisPt 8 μg/mL. C : C = HepG2 cells exposed to CisPt 8 μg/mL and carnosine in 1 : 3 molar ratio. Car = HepG2 cells exposed to carnosine. Water = HepG2 cells exposed to water (carnosine control). Saline = HepG2 cells exposed to saline (CisPt control). W : S = HepG2 cells exposed to water and saline (C : C control). (Error bars represent the standard deviation.) The P values on the bars denote the significant differences as compared to the control cells that were seeded in a stationary plate and with media not supplemented with any chemical agent (^*** P < 0.001).

Figure 7

Schematic diagram for the effect of CisPt, mechanical stress, and CisPt combined with mechanical stress on HepG2 cells. (a) HepG2 cells without exposure to any stress factors have reactive oxygen species (ROS) bound to glutathione or to carnosine. (b) Upon addition of CisPt, low cell death results probably due to the complexation of some CisPt molecules to carnosine or glutathione in the cytoplasm. (c) Upon exposure of HepG2 cells to mechanical stress, complexes of ROS-glutathione and ROS-carnosine are disassembled; hence ROS is free to enter the nucleus and cause moderate cell death. (d) In the presence of CisPt and mechanical stress, many CisPt-Car, CisPt-glutathione, ROS-carnosine, and ROS-glutathione complexes are disassembled and many CisPt and ROS molecules enter the nucleus resulting in high cell death.