Chemoresistance to Cancer Treatment: Benzo-α-Pyrene as Friend or Foe?

Abstract

Background: Environmental pollution such as exposure to pro-carcinogens including benzo-α-pyrene is becoming a major problem globally. Moreover, the effects of benzo-α-pyrene (BaP) on drug pharmacokinetics, pharmacodynamics, and drug resistance warrant further investigation, especially in cancer outpatient chemotherapy where exposure to environmental pollutants might occur. Method: We report here on the effects of benzo-α-pyrene on esophageal cancer cells in vitro, alone, or in combination with chemotherapeutic drugs cisplatin, 5-flurouracil, or paclitaxel. As the study endpoints, we employed expression of proteins involved in cell proliferation, drug metabolism, apoptosis, cell cycle analysis, colony formation, migration, and signaling cascades in the WHCO1 esophageal cancer cell line after 24 h of treatment. Results: Benzo-α-pyrene had no significant effect on WHCO1 cancer cell proliferation but reversed the effect of chemotherapeutic drugs by reducing drug-induced cell death and apoptosis by 30−40% compared to drug-treated cells. The three drugs significantly reduced WHCO1 cell migration by 40−50% compared to control and BaP-treated cells. Combined exposure to drugs was associated with significantly increased apoptosis and reduced colony formation. Evaluation of survival signaling cascades showed that although the MEK-ERK and Akt pathways were activated in the presence of drugs, BaP was a stronger activator of the MEK-ERK and Akt pathways than the drugs. Conclusion: The present study suggest that BaP can reverse the effects of drugs on cancer cells via the activation of survival signaling pathways and upregulation of anti-apoptotic proteins such as Bcl-2 and Bcl-xL. Our data show that BaP contribute to the development of chemoresistant cancer cells.

Keywords: 5-fluorouracil; apoptosis; benzo-α-pyrene; chemoresistance; cisplatin; drug metabolizing enzymes; environmental pollution; esophageal cancer; paclitaxel; procarcinogen.

Figure 1

Scheme of WHCO1 and WHCO5 cancer cell treatment (A) Evaluation of effect of benzo-α-pyrene (BaP) on the response of WHCO1 and WHCO5 cancer cells to individual drugs; (B) Evaluation of effect of BaP on the response of WHCO1 and WHCO5 cancer cells to combinations of drugs.

Figure 2

Benzo-α-pyrene abrogates drug-induced-apoptosis and colony formation inhibition. (A) WHCO1 cells (3 × 10⁵) were plated in 6-well plates overnight. WHCO1 cells were then treated with 0.1% DMSO, 4.2 µM CDDP, 3.5 µM 5-FU, 2 µM paclitaxel, and 10 µM BaP for 24 h. Cells were double stained with Annexin V and Propidium Iodide and analyzed by flow cytometry to detect apoptosis; (B) Quantification of apoptosis after treatment of WHCO1 cells as described in (A) based on the percentage of cells in each quadrant (Q1, Q2, Q3, Q4); (C) WHCO1 cells (1 × 10³) were plated in 6-well plates overnight. WHCO1 cells were then treated with 0.1% DMSO, 4.2 µM CDDP, 3.5 µM 5-FU, 2 µM paclitaxel, and 10 µM BaP and incubated for 8 days. After 8 days, colonies were fixed with 4% para-formaldehyde, stained with 0.1% crystal violet, and counted; (D) Quantification of colonies after treatment of WHCO1 cells as described in (C). * p < 0.05.

Figure 3

Benzo-α-pyrene differentially influence the expression of CYP1A1, CYP1A2, CYP1B1, and GSTP1 in WHCO1 in response to chemotherapeutic drugs. WHCO1 cells (5 × 10⁵) were plated in 6-well plates overnight. WHCO1 cells were then treated with 0.1% DMSO, 3.5 µM 5-FU, 4.2 µM cisplatin, 2 µM paclitaxel, and 10 µM BaP for 6, 12, and 24 h. Cells were lysed with RIPA buffer and proteins quantified using the BCA protein quantification assay. (A) Immunoblot analysis of proteins extracted from WHCO1 cells treated with 5-FU and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies; (B) Immunoblot analysis of proteins extracted from WHCO1 cells treated with cisplatin and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies; (C) Immunoblot analysis of proteins extracted from WHCO1 cells treated with paclitaxel and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies. GAPDH was used as a loading control.

Figure 4

Benzo-α-pyrene reverses the dual effect of cisplatin, 5-FU, and paclitaxel on WHCO1 apoptosis. WHCO1 cells (5 × 10⁵) were plated in 6-well plates overnight. WHCO1 cells were then treated with 0.1% DMSO, 4.2 µM CDDP, 3.5 µM 5-FU, 2 µM paclitaxel, 10 µM BaP, and their combinations for 24 h. Cells were double stained with Annexin V and Propidium Iodide and analyzed by flow cytometry to detect apoptosis. (A) Flow cytometric analysis of WHCO1 cells after treatment with 0.1% DMSO, 4.2 µM cisplatin, 3.5 µM 5-FU, 2 µM, 10 µM BaP, and their combinations; (B) Quantification of apoptosis after treatment of WHCO1 cells as described in (A) based on the percentage of cells in each quadrant (Q1, Q2, Q3, Q4). * p < 0.05.

Figure 5

Benzo-α-pyrene reverses the dual effect of cisplatin, 5-FU, and paclitaxel on WHCO1 colony formation. WHCO1 cells (1 × 10³) were plated in 6-well plates overnight. WHCO1 cells were then treated with 0.1% DMSO, 4.2 µM CDDP, 3.5 µM 5-FU, 2 µM paclitaxel, 10 µM BaP, and their combinations. Cells were cultured for another 8 days. Para-formaldehyde (4%) was used to fix the cells and staining was done using 0.1% crystal violet. (A,C,E) Representative images of colonies formed when cells were treated with 5FU, CDDP, Paclitaxel, BaP and their combinations; (B,D,F) Colonies were counted using the UVP software and the relative numbers were plotted on a graph. * p < 0.05.

Figure 6

Benzo-α-pyrene abrogates the effect of cisplatin, 5-FU, paclitaxel, and their combinations on WHCO1 cell migration. WHCO1 cells (5 × 10⁵) were plated in 6-well plates until confluent. Scratch wounds were made using a 200 ul pipette tip and cells were treated with 0.1% DMSO, 4.2 µM CDDP, 3.5 µM 5-FU, 2 µM, 10 µM BaP, and their combinations 24 h. At indicated time points during incubation, images of the scratch wounds were taken using a Phase Contrast inverted microscope (Olympus CKX41). (A) Effect of BaP on WHCO1 cell migration in response to cisplatin, 5-FU, and their combinations; (B) Effect of BaP on WHCO1 cell migration in response to 5-FU, paclitaxel, and their combinations; (C) Effect of BaP on WHCO1 cell migration in response to cisplatin, paclitaxel, and their combinations. Results are shown as an average of three independent experiments.

Figure 7

Benzo-α-pyrene differentially affect the expression of CYP1A1, CYP1A2, CYP1B1, and GSTP1 in WHCO1 cells in the presence of drugs. WHCO1 cells (5 × 10⁵) were plated in 6-well plates overnight. WHCO1 cells were then treated with 0.1% DMSO, 4.2 µM, 3.5 µM 5-FU, 2 µM paclitaxel, and 10 µM BaP for 24 h. (A) RT PCR analysis was performed using CYP1A1, CYP1A2, CYP1B1, and GSTP1 primers after treatment with a combination of cisplatin, 5-fluorouracil, and BaP; (B) RT PCR analysis was performed after treatment with a combination of 5-fluorouracil, paclitaxel, and BaP; (C) RT PCR analysis was performed after treatment with a combination of cisplatin, paclitaxel, and BaP. GAPDH was used as a normalizer. * p < 0.05.

Figure 8

Benzo-α-pyrene reverse the effect of drugs on Akt and MEK-ERK signaling pathways in WHCO1 cells. WHCO1 cells (5 × 10⁵) were plated in 6-well plates overnight. WHCO1 cells were then treated with 0.1% DMSO, 4.2 µM cisplatin, 3.5 µM 5-FU, 2 µM, and 10 µM BaP for 24 h. Cells were lysed with RIPA buffer and proteins quantified using the BCA protein quantification assay. (A) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with cisplatin, 5-fluorouracil, and BaP; (B) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with 5-fluorouracil, paclitaxel, and BaP; (C) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with cisplatin, paclitaxel, and BaP.