An organotin indomethacin derivative inhibits cancer cell proliferation and synergizes the antiproliferative effects of lapatinib in breast cancer cells

Abstract

It is known that an inflammatory condition in different types of cancer provides a sustained microenvironment that favors tumor growth, invasion, and metastasis. Non-steroidal anti-inflammatory drugs such as indomethacin have demonstrated chemo-preventive, anti-proliferative and cytotoxic effects in a variety of tumors. The aim of this study was to investigate the effects of an organotin indomethacin derivative (OID) on the proliferation of breast and prostate cancer cell lines and the possible mechanisms of action of this compound. Different cancer cell lines were treated in the presence of OID and cell proliferation was measured by quantification of the DNA content, changes in the cell cycle profile and the activation of caspase 3 were evaluated by flow cytometry, interleukin 6 (IL-6) gene expression was evaluated by qPCR and protein expression was analyzed by ELISA and Western blot assays. OID inhibited the cell proliferation of a panel of cancer cell lines in a concentration-dependent manner. Moreover, the addition of OID to lapatinib treatment, targeted therapy for breast cancer, significantly enhanced its antiproliferative response. The effects on cell proliferation of these compounds involved, among others, the induction of apoptosis, the downregulation of IL-6 and a decrease of the MAPK activation pathway. Our results suggest that the use of OID alone or in combination with tyrosine kinase inhibitors could be considered as adjuvants in the treatment of cancer.

Keywords: IL-6; Organotin derivative indomethacin; apoptosis; cancer cell proliferation; lapatinib.

Figure 1

Antiproliferative effect of OID in cancer cells. (A) Breast (SUM-229PE (SUM), SK-BR3 (SK), HCC1937 (HCC)), (B) prostate (DU-145 (DU), PC-3), (C) cervical (HeLa) cancer cells, and (D) lymphocytes (lym) stimulated with PHA (phytohemagglutinin) were incubated in the presence of different OID concentrations during 5 days. Cell proliferation was evaluated by quantification of DNA. Results are shown as the mean ± S.D. of sextuplicate determinations of three independent experiments. Data from vehicle-treated cells (V) were normalized to 100%.

Figure 2

OID improved lapatinib antiproliferative effect in breast cancer cells and altered cell cycle profile in breast and prostate cancer cells. (A and B) Breast cancer cells were treated in the absence (V) or presence of OID, lapatinib (L) or their combination during 72 hrs. Also, (C and D) prostate cancer cells were incubated with OID or its vehicle. After that, cell proliferation and cell cycle profile were evaluated. Bars represent the mean ± S.D. of three independent experiments per triplicate. *P≤0.05 vs. vehicle, **P≤0.05 vs. each compound alone.

Figure 3

OID induced apoptosis in cancer cells. (A) breast and (C) prostate cancer cells were incubated in the absence (V) or presence of OID alone or in combination with lapatinib (L) during 48 h and active caspase 3-positive cells analysis was performed. The cells positive to caspase 3 are shown in the gate. Quantification of percentage of cells positive to caspase 3 of (B) breast and (D) prostate cancer cells obtained of three experiments independently. *P≤0.05 vs. V, **P≤0.05 vs. each compound alone.

Figure 4

OID alone or in combination with lapatinib treatment inhibited the MAPK signaling pathway in cancer cells. (A) Breast or (C) prostate cancer cells were incubated in the absence (V) or presence of OID alone or with lapatinib (L) during 48 h. The phosphorylated form of ERK1/2 (Thr202/Tyr204) protein was determined by Western blot. Glyceraldehyde 3-phosphate dehydrogenase protein (GAPDH) was used as the loading control. Representative image from three independent experiments is shown. Normalization was performed against to total form of ERK protein in (B) SUM-229PE and (D) DU-145 cancer cells. Densitometry of three independent experiments is shown. *P≤0.05 vs. V.

Figure 5

OID inhibited the expression of pro-inflammatory cytokines. Breast (SUM-229PE) and prostate (DU-145) cancer cells were incubated in the absence (V) or presence of OID alone or with lapatinib (L) during 24 or 48 h. Relative gene expression of IL-6 (A and B) were determined by RT-PCR. Glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) was considered as constitutive reference. For IL-6 (C and D) protein expression, the supernatants of cell cultures treated were quantified by ELISA. *P≤0.05 vs. V.