Elsevier

Radiotherapy and Oncology

Volume 92, Issue 3, September 2009, Pages 339-344
Radiotherapy and Oncology

Molecular radiobiology
Inhibition of STAT-3 results in radiosensitization of human squamous cell carcinoma

https://doi.org/10.1016/j.radonc.2009.06.022Get rights and content

Abstract

Background

Signal transducer and activator of transcription-3 (STAT-3) is a downstream component of the Epidermal Growth Factor Receptor (EGFr) signaling process that may facilitate the resistance of tumor cells to conventional cancer treatments. Studies were performed to determine if inhibition of this downstream protein produces radiosensitization.

Methods/Results

A431 cells (human squamous cell carcinoma cells with EGFr overexpression) were found to be sensitized to radiation after treatment with STAT-3 small interfering RNA (siRNA). Therefore, a short hairpin RNA (shRNA) against STAT-3 was designed and cloned into a pBABE vector system modified for shRNA expression. Following transfection, clone 2.1 was selected for further study as it showed a dramatic reduction of STAT-3 protein (and mRNA) when compared to A431 parental cells or a negative control shRNA cell line (transfected with STAT-3 shRNA with 2 base pairs mutated). A431 2.1 showed doubling times of 25–31 h as compared to 18–24 h for the parental cell line. The A431 shRNA knockdown STAT-3 cells A431 were more sensitive to radiation than A431 parental or negative STAT-3 control cells.

Conclusion

A431 cells stably transfected with shRNA against STAT-3 resulted in enhanced radiosensitivity. Further work will be necessary to determine whether the inhibition of STAT-3 phosphorylation is a necessary step for the radiosensitization that is induced by the inhibition of EGFr.

Section snippets

Cell culture

A431 human epidermoid cancer cells were obtained from American Type Culture Collection (Manassas, VA) and processed for cell culture as described in the previous studies from our laboratory [14]. Briefly, the cell lines were maintained in Dulbeco’s modified Eagle’s medium: F12 (50:50) containing 7% fetal bovine serum supplemented with l-glutamine and were incubated at 37 °C in 5% CO2.

Cell proliferation

This assay of daily growth was performed as previously described [10], [12]. Following the plating of A431 cells,

Results

Since activated STAT-3 is an important regulator of tumor growth and portends resistance to apoptosis in tumor cells, it was hypothesized that the inhibition of STAT-3, through the use of small interfering RNA (siRNA), may potentiate the effects of radiation in A431 human squamous cell carcinoma cells. In fact, when cells were transfected with 100 nM siRNA, using Oligofectamine, for as little as 4 h prior to various doses of radiation, the cells were significantly more responsive to the cytotoxic

Discussion

Although constitutive activation of STAT-3 has been shown to be characteristic of many tumors [6], [7], additional studies have shown that STAT-3 is greatly influenced by several upstream regulators of tumor growth [8], [11]. For instance, STAT-3 plays an important role in transmitting the growth signals that are initiated at the level of EGFr [3], [4], [5], [8]. EGFr is also known to be overexpressed in many tumors and the inhibition of EGFr has been shown to enhance the effects of

Acknowledgement

This study was supported in part by NIH R01 CA89297.

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