Adenylate kinase 3 sensitizes cells to cigarette smoke condensate vapor induced cisplatin resistance

Abstract

Background: The major established etiologic risk factor for bladder cancer is cigarette smoking and one of the major antineoplastic agents used for the treatment of advanced bladder cancer is cisplatin. A number of reports have suggested that cancer patients who smoke while receiving treatment have lower rates of response and decreased efficacy of cancer therapies.

Methodology/principal findings: In this study, we investigated the effect of cigarette smoke condensate (CSC) vapor on cisplatin toxicity in urothelial cell lines SV-HUC-1 and SCaBER cells. We showed that chronic exposure to CSC vapor induced cisplatin resistance in both cell lines. In addition, we found that the expression of mitochondrial-resident protein adenylate kinase-3 (AK3) is decreased by CSC vapor. We further observed that chronic CSC vapor-exposed cells displayed decreased cellular sensitivity to cisplatin, decreased mitochondrial membrane potential (ΔΨm) and increased basal cellular ROS levels compared to unexposed cells. Re-expression of AK3 in CSC vapor-exposed cells restored cellular sensitivity to cisplatin. Finally, CSC vapor increased the growth of the tumors and also curtail the response of tumor cells to cisplatin chemotherapy in vivo.

Conclusions/significance: The current study provides evidence that chronic CSC vapor exposure affects AK3 expression and renders the cells resistant to cisplatin.

Figure 1. CSC vapor decreased the expression of AK3 and increased cisplatin resistance.

(a) Western blot analysis was performed using anti-AK3 and anti-AK2 antiserum. Protein extracts in each lane are as indicated. Even loading was confirmed by re-probing the membrane with β-Actin antibody. HUC1-6M or SCaBER-6M indicates the cells chronically exposed to CSC vapor. HUC1-0.1%6M are SV-HUC-1 cells exposed to 0.1% CSC for 6 months. SV-HUC-1 and HUC1-6M (b), SCaBER and SCaBER-6M (c) were treated with 0 to 5 µg/ml cisplatin for 48 h, or with 3 µg/ml cisplatin for 24, 48 and 72 h, in the presence and absence of AK3 as indicated. (d) Colony formation assays were performed with SCaBER and SCaBER-6M in the presence and absence of AK3 as indicated. Data were expressed as mean ±SD. * indicates P<0.05 (e) SV-HUC-1 and SCaBER cells were transfected with AK3 RNAi for 48 h and Western blot analysis was performed using anti-AK3 antiserum. (f) SCaBER cells were transfected with AK3 RNAi and 48 h after transfection the cells were treated with 0 to 5 µg/ml cisplatin for 48 h and cellular survival was assessed. Error bars represent standard deviation of three experiments.

Figure 2. ΔΨm Alteration by AK3 in the Presence of Cisplatin.

Mitochondrial membrane potential was measured in SV-HUC1, HUC1-6M (a), SCaBER and SCaBER-6M (b) cells transfected with AK3 or control vector, followed by treatment with cisplatin for 48 h. Cells were stained with JC-1 reagent and cell fluorescence was measured on a flow cytometer using Fl1 and Fl2 channels. Increase of red fluorescence indicates hyper-polarization of mitochondrial membrane potential (ÄΨm). * indicates P<0.05 and ** indicates P<0.01.

Figure 3. ROS Production by AK3 in the Presence of Cisplatin.

ROS production was measured in parental SV-HUC-1 and SCaBER cells or cells exposed to CSC vapor for 6 months using DCFH-DA staining. The cells were transfected with or without the AK3 expression plasmid and treated with or without cisplatin for 48 h before staining. ROS generation in SV-HUC-1 and HUC1-6M (a–c) and SCaBER and SCaBER-6M (d–f) cells with overexpression of AK3 with and without cisplatin. The mean fluorescence density was calculated and data was plotted as mean ± SD (g,h).

Figure 4. AK3 Induced Release of Lactate Dehyderogenase upon Treatment of Cisplatin.

LDH release in SV-HUC-1 (a) and SCaBER (b) cells exposed to CSC vapor in presence of AK3 followed by cisplatin treatment for 48 h. The data are presented as means ± SD. All experiments were performed in triplicate. Statistical significance is as indicated with * indicates P<0.05 and ** indicates P<0.01

Figure 5. Activation of Apoptosis in the Presence of AK3 and Cisplatin.

Apoptosis was measured in the SV-HUC-1 and HUC1-6M cells using annexin/PI staining. The cells were transfected with or without AK3 expression plasmid and treated with or without cisplatin for 48 h as indicated.

Figure 6. CSC vapor induces Survival Proteins.

(a) Cellular lysates were obtained from the indicated cell lines and subjected to Western Blot analysis using the indicated antibodies. β-actin was used as a control. (b) The indicated cell lines were treated with 3 µg/ml cisplatin for 48 h. Mitochondrial and cytoplasmic total cellular lysates were subjected to Western Blot analysis using anti-cytochrome c antibody. Protein extracts in each lane are as indicated. Immunodetection of â-actin and COX II were done to assure that the transfected proteins were in cytoplasm and mitochondria respectively.

Figure 7. CSC Vapor Induces Cisplatin Resistance In Xenograft Model.

(a) Subcutaneous xenografts were generated in of 5- to 6-week-old female athymic nu/nu mice with SCaBER or SCaBER-6M cells. One week following tumor cell inoculation, mice were treated with either PBS or cisplatin (3 mg/Kg/dose). The mean tumor volume was calculated. * indicates P<0.01 (b) One representative mouse from each group was displayed.