Ammonium tetrathiomolybdate enhances the antitumor effect of cisplatin via the suppression of ATPase copper transporting beta in head and neck squamous cell carcinoma

Abstract

Platinum‑based antitumor agents have been widely used to treat head and neck squamous cell carcinoma (HNSCC) and numerous other malignancies. Cisplatin is the most frequently used platinum‑based antitumor agent, however drug resistance and numerous undesirable side effects limit its clinical efficacy for cancer patients. Cancer cells discharge cisplatin into the extracellular space via copper transporters such as ATPase copper transporting beta (ATP7B) in order to escape from cisplatin‑induced cell death. In the present study, it was demonstrated for the first time that the copper chelator ammonium tetrathiomolybdate (TM) has several promising effects on cisplatin and HNSCC. First, TM suppressed the ATP7B expression in HNSCC cell lines in vitro, thereby enhancing the accumulation and apoptotic effect of cisplatin in the cancer cells. Next, it was revealed that TM enhanced the antitumor effect of cisplatin in HNSCC cell tumor progression in a mouse model of bone invasion, which is important since HNSCC cells frequently invade to facial bone. Finally, it was demonstrated that TM was able to overcome the cisplatin resistance of a human cancer cell line, A431, via ATP7B depression in vitro.

Figure 1.

Expression of ATP7B in head and neck normal tissues, head and neck cancers, and human oral squamous cell lines. (A) Immunohistochemistry results of ATP7B expression in (a) head and neck normal tissue and (b) HNC. (B) Scatterplot of the ATP7B-positive areas in the head and neck normal tissues (n=11) and HNSCC (n=70). Error bars: Mean ± SD. There was a significantly increased expression of ATP7B in the HNSCC samples (P<0.0001). (C) The expression of ATP7B in the human oral squamous cell lines (HSC-2, −3, −4) analyzed by western blotting. HNC, head and neck cancer; HNSCC, head and neck squamous cell carcinoma; ATP7B, ATPase copper transporting beta.

Figure 2.

Effect of TM on ATP7B expression and the synergistic antitumor effect of cisplatin in HNSCC cell proliferation in vitro via cisplatin accumulation. HSC3 cells were plated in triplicate and treated with 5 µM TM for 24 h. (A) The expression of ATP7B in control HSC-3 cells and TM-treated HSC-3 cells was analyzed based on the image blot density revealed by western blotting. (B) Immunofluorescence analysis of ATP7B and DAPI in the cells. Upper images, ATP7B (green); middle images, DAPI (blue); lower images, merged. Sections were incubated with rabbit anti-ATP7B (1:100), then with Alexa Fluor 488 anti-rabbit IgG (1:1,000) and encapsulated with DAPI. (C) The cisplatin concentration in HSC-3 cells was evaluated after 6-h cisplatin treatment with or without 24-h TM pre-incubation. (D) Antitumor and synergistic antitumor effect of TM against HSC-3 HNSCC cells. HSC-3 cells were treated with cisplatin with or without TM for 24 h. Live cells were counted with trypan blue reagent. Data represent the relative ratio (the control is indicated as 1.0). *P<0.05 cisplatin + TM group vs. the cisplatin treated group. TM, tetrathiomolybdate; ATP7B, ATPase copper transporting beta; HNSCC, head and neck squamous cell carcinoma.

Figure 3.

Apoptosis effects of TM and cisplatin on HNSCC cells grown in vitro. (A) The expression of total and cleaved caspase-3 in HSC-3 cells treated with or without cisplatin and TM. Total and cleaved caspase-3 in HSC-3 cells was evaluated based on the image blot density revealed by western blotting. (B) Immunofluorescence analysis of Tunnel and DAPI in the HSC-3 cells. Upper images, Tunnel (green); middle images, DAPI (blue), lower images, merged. (C) Flow cytometric analysis: Propidium iodide (PI) and Annexin V staining of HSC-3 cells treated with cisplatin or with cisplatin+TM for 24 h. Apoptopic cells were evaluated with FACS Aria III. TM, tetrathiomolybdate; HNSCC, head and neck squamous cell carcinoma. Data are expressed as the mean ± SD. *P<0.05. Cisplatin+ TM-treated vs. cisplatin-treated cells.

Figure 4.

Effects of tetrathiomolybdate (TM) on the mouse model of cancer-associated bone destruction. (A) Representative radiographs and micro-CT of a tibia from a mouse with HSC-3 injected bone invasion after a single treatment of TM or low-dose cisplatin or a combination treatment of TM+low-dose cisplatin. (B) Bone resorption area with a soft X-ray photograph image, assessed with the LuminaVision imaging software program (Mitani Corporation). Data are expressed as the mean ± SD. *P<0.05. Cisplatin+ TM-treated vs. cisplatin-treated mice. (C) Histological and histomorphometric analysis of the bone marrow in the bone invasion mouse model. The sections of mouse tibial bone marrow were stained with hematoxylin and eosin. (D) Osteoclast visualized with TRAP stain. TM, tetrathiomolybdate; H&E, hematoxylin and eosin; TRAP, tartrate-resistant acid phosphatase.

Figure 5.

Immunofluorescence analysis of the tumor in the bone marrow in the mouse model of bone invasion. Representative sections of HSC-3 injected mouse tibial bone marrow stained with (A, low magnification) H&E and DAPI; (B, green: High magnification) cleaved caspase-3 and DAPI stained with blue (n=6/group). H&E, hematoxylin and eosin.

Figure 6.

Immunofluorescence analysis of the tumor in the bone marrow in the mouse model of bone invasion. (A, green: High magnification) KI-67, (B, green: High magnification) ATP7B and DAPI stained with blue (n=6/group). ATP7B, ATPase copper transporting beta.

Figure 7.

The role of ATP7B in cisplatin-resistant cancer cells. (A) The expression of ATP7B in the parental and cisplatin-resistant A431 cancer cells (A431/CDDP-R) was evaluated by a western blot analysis. (B) A431 and A431/CDDP-R cells were incubated with cisplatin with or without TM. Cleaved caspase-3 was evaluated with western blotting. TM, tetrathiomolybdate; ATP7B, ATPase copper transporting beta; CDDP, cis-dichloro-diamine-platinum-resistant.