Involvement of endoplasmic reticulum stress and cell death by synthesized Pa-PDT in oral squamous cell carcinoma cells

Abstract

Background/purpose: Photodynamic therapy (PDT) is a therapeutic alternative for malignant tumors that uses a photosensitizer. This study examined whether synthesized Pheophorbide a (Pa) -PDT induced apoptosis and autophagy involving endoplasmic reticulum (ER) stress in oral squamous cell carcinoma (OSCC) cells.

Materials and methods: Human OSCC cells were treated with Pa-PDT, and cell proliferation was examined by MTT assay. Apoptosis and autophagy were measured using Western blot analysis. ER stress was examined using RT-PCR and Western blot analysis. In vivo murine OSCC animal model were treated with intratumoral (IT) Pa-PDT, and investigated the therapeutic effect.

Results: Pa-PDT significantly inhibited the proliferation of human OSCC cells in a dose-dependent manner. Pa-PDT induced intrinsic apoptotic cell death and also induced autophagy. Pa-PDT induced ER stress which was observed as demonstrated by the up-regulation of the ER stress marker. Inhibition of the ER stress pathway using 4-phenylbutyric acid (PBA) decreased CHOP and induced inhibition of cell deaths. In addition, the inhibition of ER stress enhanced Pa-PDT mediated autophagy. IT Pa-PDT significantly inhibited the tumor growth and induced apoptosis, autophagy and ER stress in vivo OSCC cells transplanted model.

Conclusion: This study showed that synthesized Pa-PDT induced ER stress trigger apoptosis and apoptotic cell death pathways in OSCC cells. The inhibition of ER stress declined Pa-PDT mediated cytotoxicity with an increase of autophagy. These results may provide Pa-PDT exerts anti-tumor effects through ER stress pathway in OSCC cells and may provide a basis for developing Pa-PDT targeting ER stress as a therapy for OSCC.

Keywords: Apoptosis; Autophagy; ER stress; Oral sqaumous cell carcinoma; Pa-PDT.

Figure 1

The effects of Pa-PDT on proliferation in human OSCC cells. (A and B) FaDu and YD-10B cells were preincubated with various Pa concentrations (0.05–0.5 μM) for 2 h and then illuminated (1.25 J/cm²). The levels of cell proliferation were measured using an MTT assay at 24h after Pa-PDT treatment. The percentage of viable cells was calculated as the ratio of treated cells to the control cells. The data are reported as the mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with the control group.

Figure 2

Apoptosis and autophagy analysis of Pa-PDT treated human OSCC cells. (A) Expression of apoptosis-related proteins in Pa-PDT treated cells. (B) Expression of autophagy-related proteins in Pa-PDT treated cells. FaDu and YD-10B cells were treated with Pa-PDT for 24 h at indicated concentration. The total cell lysates were prepared and the protein was subjected to SDS–PAGE followed by Western blot analysis and chemiluminescent detection. Western blot analysis was performed using a series of antibodies; Bcl-2, caspase-7, cleaved caspase-3, PARP, Beclin1, ATG5, ATG12 and LC3B. The protein levels were normalized by a comparison to the actin levels.

Figure 3

Induction of ER stress in Pa-PDT-treated human OSCC cells. (A) The mRNA expression levels of CHOP in Pa-PDT treated cells. mRNA was isolated and RT-PCR was performed. The PCR products were separated on 1.5% agarose gels and values were normalized by comparing to the GAPDH. (B) Expression of ER stress-related proteins in Pa-PDT treated cells. (C) Expression of ER stress-induced cell death-related proteins in Pa-PDT treated cells. FaDu and YD-10B cells were treated with Pa-PDT for 24 h at indicated concentration. Cell lysates were subjected to Western blot analysis using antibodies; CHOP, caspase-12, p-IRE1α, p-PERK, ATF6 and Bip. The protein levels were normalized by comparing to the actin levels.

Figure 4

Inhibition of ER stress by 4-phenylbutyrate (PBA) in Pa-PDT-treated human OSCC cells. The cells were treated with Pa-PDT for 24 h in the presence or absence of PBA (1 mM). (A) The mRNA expression levels of CHOP in Pa-PDT and PBA co-treated cells. mRNA was isolated and RT-PCR was performed. The PCR products were separated on 1.5% agarose gels and values were normalized by comparing to the GAPDH. (B) Western blot analysis was performed using ER stress-related proteins antibodies (CHOP, caspase-12) in cells co-treated with Pa-PDT with PBA. The protein levels were normalized by a comparison with the actin levels. The relative ratio in expressed protein was assessed by scanning densitometry. The data are reported as the mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with the Pa-PDT treated group. (C) Cell viability was determined by MTT assay after co-treated with Pa-PDT and PBA. The percentage of viable cells was calculated as the ratio of treated cells to the control cells. The data are reported as the mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with the Pa-PDT treated group.

Figure 5

Regulation of apoptosis and autophagy by the inhibition of ER stress in Pa-PDT-treated human OSCC cells. The cells were treated with Pa-PDT for 24 h in the presence or absence of PBA (1 mM). (A) Expression of apoptosis-related proteins in cells co-treated with Pa-PDT with PBA. (B) Conversion of LC3B, the autophagy marker protein after co-treated with Pa-PDT and PBA. Cell lysates were subjected to Western blot analysis using antibodies; cleaved caspase-7, PARP and LC3B. The protein levels were normalized by a comparison with the actin levels. The relative ratio in expressed protein was assessed by scanning densitometry. The data are reported as the mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with the Pa-PDT treated group. (C) The effect of PBA on Pa-PDT-induced acidic vesicular organelle (AVO) formation. After Pa-PDT treatment with PBA, acridine orange (1 μg/ml) was added to the living cells for 30 min and the cells were visualized under a fluorescence microscope (200 × magnification).

Figure 6

Effect of IT Pa-PDT on tumor growth, apoptosis, autophagy and ER stress in vivo model. C3H mice bearing AT-84 murine oral cancer cells were treated with IT Pa-PDT. (A) Relative tumor volumes of the mice treated with IT Pa-PDT and the vehicle-treated controls. Tumor volume was measured every 2 days and calculated by the formula: V = (ab²)/2, in which a is the longest diameter, and b is the shortest diameter of the tumor. The data are reported as the mean ± SD of five animals. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with the control group. (B) H&E staining, immunohistochemistry for PCNA, TUNEL assay were performed on paraffin sections from the tumor. Immunohistochemical study for autophagy (C) and ER stress (D) were performed on paraffin sections from the tumor using antibodies; LC3B, Beclin1, p62, CHOP, p-PERK, ATF6. Photographs were taken under a magnification of 400 × .