Antineoplastic and apoptotic potential of traditional medicines thymoquinone and diosgenin in squamous cell carcinoma

Abstract

Thymoquinone (TQ) and diosgenin (DG), the active ingredients obtained from black cumin (Nigella sativa) and fenugreek (Trigonella foenum graecum), respectively, exert potent bioactivity, including anticancer effects. This study investigated the antineoplastic activity of these agents against squamous cell carcinoma in vitro and sarcoma 180-induced tumors in vivo. TQ and DG inhibited cell proliferation and induced cytotoxicity in A431 and Hep2 cells. These agents induced apoptosis by increasing the sub-G(1) population, LIVE/DEAD cytotoxicity, chromatin condensation, DNA laddering and TUNEL-positive cells significantly (P<0.05). Increased Bax/Bcl-2 ratio, activation of caspases and cleavage of poly ADP ribose polymerase were observed in treated cells. These drugs inhibited Akt and JNK phosphorylations, thus inhibiting cell proliferation while inducing apoptosis. In combination, TQ and DG had synergistic effects, resulting in cell viability as low as 10%. In a mouse xenograft model, a combination of TQ and DG significantly (P<0.05) reduced tumor volume, mass and increased apoptosis. TQ and DG, alone and in combination, inhibit cell proliferation and induce apoptosis in squamous cell carcinoma. The combination of TQ and DG is a potential antineoplastic therapy in this common skin cancer.

Figure 1. Dose-dependent growth inhibition of squamous SCC cells by thymoquinone (TQ) and diosgenin (DG).

(A) A431, Hep2 and RPMI 2650 cells were treated with different concentrations of TQ or DG for 48 hours and MTT assays were performed. Points represent means ± SEM, n = 8 in three different experiments. (B) Phase-contrast photomicrographs (Leica 4×) of A431, Hep2 and HaCaT cells treated with different concentrations of TQ or DG for 48 hours.

Figure 2. TQ- and DG- induce apoptosis in A431 and Hep2 cells.

(A) Dose-dependent effects of TQ and DG detected by cell cycle–based apoptosis assay. Cells were treated with respective IC₅₀ of TQ or DG for 48 hours, stained with PI and measured by flow cytometry. (B) Accumulation of sub-G₁ cells plotted from the cell cycle–based apoptosis assay. Data are presented as means ± SEM from three independent experiments. Dissimilar superscripts (a, b, c, d) represent the significant differences among them (P<0.05). (C) After treating with IC₅₀ of TQ or DG, cells were stained with Annexin V-FITC and PI and analysed by fluorescence microscopy. Three primary populations of cells: cells are viable and not undergoing apoptosis (Annexin V-FITC and PI negative), cells undergoing apoptosis (Annexin V-FITC positive and PI negative) and cells in end-stage apoptosis or already dead (Annexin V-FITC and PI positive).

Figure 3. Fluorescence-based cytotoxicity assay and chromatin condensation analysis.

(A) Fluorescence-based cytotoxicity assay (using the LIVE/DEAD Viability/Cytotoxicity kit) of A431 and Hep2cells treated with TQ or DG for 24 or 48 hours. A significantly higher percentage of apoptotic cells (live (green)/dead (red)) are observed in the treated groups than in controls. (B) Fluorescence microscopic observation of A431 and Hep2 treated with IC₅₀ of TQ and DG for 24 or 48 hours and stained with DAPI to detect chromatin condensation.

Figure 4. TQ- and DG- induced apoptosis as measured by DNA laddering and TUNEL assay.

(A) Representative image of TUNEL assay in TQ- or DG- treated A431 and Hep2 cells. (B) Apoptotic cells are quantified by counting the percentages of TUNEL-positive nuclei. Data are presented as means ± SEM of three independent experiments. Dissimilar superscripts (a, b, c, d) represent the significant differences among them (P<0.05). (C) DNA laddering of A431 and Hep2 cells treated with IC₅₀ of TQ or DG for 48 hours.

Figure 5. TQ- and DG- induced apoptosis by altering the apoptotic proteins and signalling molecules.

(A) Western blot analysis of proapoptotic and antiapoptotic proteins in A431 and Hep2 cells treated with TQ or DG for various durations. (B) Western blot analysis of phosphorylated Akt and JNK during TQ- and DG-induced apoptosis. Beta-actin are used as loading control.

Figure 6. Dose effects on cell proliferation in studies of TQ and DG combinations.

(A) MTT assay of proliferation of cells treated with different concentrations of TQ and DG for 48 hours. Points represent means ± SEM of three different experiments. (B) Combination index (CI) was calculated; CI<1 suggests synergism between TQ and DG in A431 and Hep2 cells. Red crosses (1–4) represent the concentrations of TQ and DG in various combinations: 1 = 10 µM∶10 µM (TQ∶DG); 2 = 10 µM∶20 µM (TQ∶DG); 3 = 20 µM∶10 µM (TQ∶DG); and 4 = 20 µM∶20 µM (TQ∶DG).

Figure 7. Combination of TQ and DG enhances these agents' antiproliferative and apoptotic effects in SCC cells.

(A) Flow cytometry–based detection of apoptotic cell death in A431 and Hep2 cells treated with a combination of TQ 10 µM and DG 20 µM. (B) Accumulation of sub-G₁ phase apoptotic cells during treatment with the same combination of TQ 10 µM and DG 20 µM. Data represent means ± SEM; *represents significant differences between control and treated groups (P<0.05). (C) A431 and Hep2 cells treated with TQ and/or DG for 48 hours, labelled with phalloidin-TRITC (F-actin–binding protein, golden) and DAPI (DNA-binding dye, blue). TQ or DG alone effectively blocked cell-spreading lamellopodia, whereas combination treatment led to complete disorganization of actin filaments and formed F-actin fragments.

Figure 8. Antineoplastic activities of TQ and/or DG in sarcoma 180-induced xenografts in a mouse model.

Mice bearing sarcoma 180 xenografts are given tail vein injections of TQ (10 mg/kg/day) and/or DG (20 mg/kg/day) for up to 27 days. (A) Before treatment, tumors are prominent and large in both controls and treated groups. After treatment, mice are killed and their tumors are excised, measured and weighed. (B) Bar graph represents tumor masses in grams. (C) Data points represent tumor volumes for control and treatment groups. Data represent means ± SEM; * represents significant differences (P<0.05, n = 5) between control and treated groups. (D) Immunohistochemical analysis of sarcoma 180–induced tumor xenografts from mice treated with TQ and/or DG. Paraffin-embedded sections of tumors are processed and assays of TUNEL/PI and DAPI/phalloidin-TRITC and immunohistochemical assays of Ki-67 and CD-31 are performed.