Capsaicin displays anti-proliferative activity against human small cell lung cancer in cell culture and nude mice models via the E2F pathway

Abstract

Background: Small cell lung cancer (SCLC) is characterized by rapid progression and low survival rates. Therefore, novel therapeutic agents are urgently needed for this disease. Capsaicin, the active ingredient of chilli peppers, displays anti-proliferative activity in prostate and epidermoid cancer in vitro. However, the anti-proliferative activity of capsaicin has not been studied in human SCLCs. The present manuscript fills this void of knowledge and explores the anti-proliferative effect of capsaicin in SCLC in vitro and in vivo.

Methodology/principal findings: BrdU assays and PCNA ELISAs showed that capsaicin displays robust anti-proliferative activity in four human SCLC cell lines. Furthermore, capsaicin potently suppressed the growth of H69 human SCLC tumors in vivo as ascertained by CAM assays and nude mice models. The second part of our study attempted to provide insight into molecular mechanisms underlying the anti-proliferative activity of capsaicin. We found that the anti-proliferative activity of capsaicin is correlated with a decrease in the expression of E2F-responsive proliferative genes like cyclin E, thymidylate synthase, cdc25A and cdc6, both at mRNA and protein levels. The transcription factor E2F4 mediated the anti-proliferative activity of capsaicin. Ablation of E2F4 levels by siRNA methodology suppressed capsaicin-induced G1 arrest. ChIP assays demonstrated that capsaicin caused the recruitment of E2F4 and p130 on E2F-responsive proliferative promoters, thereby inhibiting cell proliferation.

Conclusions/significance: Our findings suggest that the anti-proliferative effects of capsaicin could be useful in the therapy of human SCLCs.

Figure 1. Capsaicin inhibited the proliferation of human SCLC cells in a time- and concentration-dependent manner.

(A) MTT assays show at the treatment of H69 and H82 cells with 50 µM capsaicin causes a decrease in cell viability starting from 24 hours to 72 hours. (B) The MTT assay was repeated in DMS53 and DMS114 cells. Similar results were obtained. (C) Capsaicin displays potent dose-dependent anti-proliferative activity in H69 human SCLC cells. H69 cells were serum-starved for 36 hours and then re-stimulated with 10% FBS for 18 hours in the presence or absence of the indicated doses of capsaicin. BrdU incorporation assays were performed to assess the anti-proliferative effects of capsaicin. (D) The experiment was repeated in H82, DMS114 and DMS53 human SCLC cell lines and similar results were obtained. Most interestingly, capsaicin displayed relatively little anti-proliferative activity in SAEC and NHBE normal lung epithelial cells, indicating that the anti-proliferative activity of capsaicin was specific for lung cancer cells. (E) The results obtained from the BrdU assays were verified by the measurement of PCNA levels in human SCLC cells. Capsaicin decreased the number of PCNA positive cells in all the four SCLC cell lines. (F) FACS analysis confirms that capsaicin causes G1/S arrest in H69 human SCLC cells. H69 cells were serum-starved for 36 hours and subsequently re-stimulated with 10% FBS for 18 hours (to induce S-phase entry) in the presence or absence of 50 µM capsaicin. Cells were then stained with propidium iodide and analyzed by FACS to quantitate the relative percentage of cells in G1 and S phase. The percentage of cells in G1 and S-phase of untreated cells were taken to be 100%, and the relative percentages of capsaicin-treated cells were calculated relative to the control. Capsaicin increased the percentage of cells in G1 phase and concomitantly decreased the percentage of cells in S-phase, indicating the presence of G1/S arrest in capsaicin-treated H69 cells. Values indicated by an “*” or a different letter are statistically significant.

Figure 2. The treatment with capsaicin correlated with decreased expression of E2F-responsive proliferative S-phase genes.

(A) Real-time PCR analysis indicated that capsaicin decreased the mRNA levels of cyclin E, TS, cdc25A and cdc6. (B) Western blotting analysis showed that capsaicin caused a concentration-dependent decrease in the levels of cyclin E, TS, cdc25A and cdc6 in H69 human SCLC cells. β-actin was used as the loading control for the western blotting experiments and the results were quantitated by densitometric analysis. (C) The western blotting experiment was repeated in H82, DMS53 and DMS114 human SCLC cells treated with 50 µM capsaicin and similar results were observed. Values indicated by an “*” are statistically significant.

Figure 3. Capsaicin inhibited the growth of human SCLC tumors in vivo.

(A) Chicken chorioallantoic membrane (CAM) assays showed that 50 µM capsaicin suppressed the growth of H69 tumors on chicken CAM. (B) Capsaicin suppressed the growth of established SCLC tumors in nude mice models. H69 cells were injected subcutaneously between the scapulae of nude mice. After the tumors attained a threshold volume of 100 mm³, the tumors were allowed to grow until a volume of 800 mm³, after which animals were divided into two groups. The treatment group was administered 10 mg of capsaicin/kg body weight in an AIN-76A based diet. The control group was administered with an AIN-76A based diet containing 10% corn oil (vehicle for capsaicin) only. Tumor volumes were calculated as (l X w X h)/2. (C) Tumor sections were stained with H and E (top panels) to assess cellular morphology and immunostained for PCNA to assess cell proliferation (bottom panels). Nude mice treated with 10 mg capsaicin/kg body weight displayed reduced cell proliferation as evidenced by decreased PCNA staining (bottom right panel) relative to control (bottom left panel). (D) Quantitation of PCNA positive cells indicated that the administration of capsaicin reduced cell proliferation in H69 tumors, relative to controls. (E) The apoptotic activity of capsaicin in nude mice models was measured by caspase cleavage assays of mouse tumor lysates. Tumor lysates from capsaicin treated mice displayed only a little increase in cellular apoptosis as compared to control mice. H69 lysates treated with 30 µM cisplatin for 72 hours was taken as the positive control for the assay. (F) Western blotting of tumor lysates from mice showed that capsaicin treatment decreased the expression of E2F-responsive proliferative genes namely cyclin E, TS, cdc25A and cdc6. β-actin was used as the loading control for the western blotting experiments. The results were quantitated by densitometric analysis. Values indicated by an “*” are statistically significant.

Figure 4. BrdU assays demonstrated that depletion of E2F4 ablates the anti-proliferative activity of capsaicin.

(A) H69 and DMS114 cells were transfected with the indicated E2F-siRNA as detailed in “Materials and Methods.” Eighteen hours post transfection, the cells were serum-starved for 36 hours and re-stimulated with media containing 10% FBS in the presence of 50 µM capsaicin for 18 hours. Subsequently, BrdU assays were performed to measure cell proliferation. The anti-proliferative activity of capsaicin was ablated by E2F4-siRNA but not affected by a non-targeting control-siRNA. (B) Western blotting analysis confirmed the suppression of E2F1–6 expression upon siRNA transfection. GAPDH was used as the loading control for the western blotting experiments, and the results were quantitated by densitometric analysis. (C) Western blotting experiments demonstrated that levels of E2F1–6 were decreased upon siRNA transfection in DMS114 cells. (D) BrdU assay showed that transfection of E2F1–6 siRNA without capsaicin did not affect the proliferation of cells in response to 10% FBS. This indicates that the effects of E2F-siRNAs observed in (A) are specifically mediated by capsaicin treatment. Values indicated by an “*” are statistically significant.

Figure 5. Two independent E2F4-siRNA reversed the anti-proliferative effect of capsaicin in H69 cells.

(A) The transfection of siRNA and subsequent BrdU assay was performed as described in “Materials and Methods.” (B) The results obtained by BrdU assay are confirmed with PCNA ELISA assay. Two independent E2F4-siRNA were transfected as described in “Materials and Methods.” PCNA assays were then performed to assess the levels of E2F4-siRNA on the anti-proliferative activity of capsaicin. (C) Western blotting analysis indicated that E2F4 levels are suppressed upon siRNA transfection. GAPDH was used as the loading control for the western blotting experiments, and the results were quantitated by densitometric analysis. Values indicated by an “*” are statistically significant.

Figure 6. ChIP assays showed that capsaicin induces the recruitment of E2F4 and p130 on proliferative promoters.

Serum-stimulated H69 cells contained robust amounts of proliferative E2Fs, namely E2F1, E2F2 and E2F3, associated with E2F-responsive proliferative promoters like cyclin E, TS, cdc25A and cdc6 (left panel). The treatment of H69 cells with 50 µM capsaicin causes dissociation of proliferative E2Fs and recruitment of the repressive E2F4 and p130 on these promoters (right panel). PCR for the c-Fos promoter (which is not regulated by E2F) was taken as the control for the experiment.