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. 2013 Jan 15;266(2):233-44.
doi: 10.1016/j.taap.2012.11.002. Epub 2012 Nov 9.

Vorinostat, an HDAC inhibitor attenuates epidermoid squamous cell carcinoma growth by dampening mTOR signaling pathway in a human xenograft murine model

Deepali Kurundkar  1 , Ritesh K Srivastava, Sandeep C Chaudhary, Mary E Ballestas, Levy Kopelovich, Craig A Elmets, Mohammad Athar
Affiliations

Vorinostat, an HDAC inhibitor attenuates epidermoid squamous cell carcinoma growth by dampening mTOR signaling pathway in a human xenograft murine model

Deepali Kurundkar et al. Toxicol Appl Pharmacol. .

Abstract

Histone deacetylase (HDAC) inhibitors are potent anticancer agents and show efficacy against various human neoplasms. Vorinostat is a potent HDAC inhibitor and has shown potential to inhibit growth of human xenograft tumors. However, its effect on the growth of skin neoplasm remains undefined. In this study, we show that vorinostat (2 μM) reduced expression of HDAC1, 2, 3, and 7 in epidermoid carcinoma A431 cells. Consistently, it increased acetylation of histone H3 and p53. Vorinostat (100mg/kg body weight, IP) treatment reduced human xenograft tumor growth in highly immunosuppressed nu/nu mice. Histologically, the vorinostat-treated tumor showed features of well-differentiation with large necrotic areas. Based on proliferating cell nuclear antigen (PCNA) staining and expression of cyclins D1, D2, E, and A, vorinostat seems to impair proliferation by down-regulating the expression of these proteins. However, it also induced apoptosis. The mechanism by which vorinostat blocks proliferation and makes tumor cells prone to apoptosis, involved inhibition of mTOR signaling which was accompanied by reduction in cell survival AKT and extracellular-signal regulated kinase (ERK) signaling pathways. Our data provide a novel mechanism-based therapeutic intervention for cutaneous squamous cell carcinoma (SCC). Vorinostat may be utilized to cure skin neoplasms in organ transplant recipient (OTR). These patients have high morbidity and surgical removal of these lesions which frequently develop in these patients, is difficult.

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Conflict of interest statement

Conflict of interest

The authors disclose that there is no conflict of interest.

Figures

Figure 1
Figure 1. Vorinostat inhibits on tumor growth, proliferation and apoptosis
(A) Graph showing mean tumor volume plotted against the study duration. Treatment was started day 3 of tumor cell inoculation. Significant reduction of average tumor volume in nu/nu mice treated with vehicle (n=5) or vorinostat (n=5) (*p=0.038). (B) Representative photographs of H&E (20X magnification), IHC (PCNA) expression (40X magnification) and TUNEL staining (20X magnification). (C) Western blot (WB) analysis of PCNA (**p=0.001) in control and vorinostat-treated tumors. (D) Relative densitometry analysis of PCNA WB. (E) Western blot analysis of Bax, Bcl2 and Cleaved caspase-3 protein isolated from excised xenograft tumor tissue. (F&G) Relative densitometry analysis of Bax (*p=0.041), Bcl2 (*p=0.027), Bax/Bcl2 ratio (*P=0.010) and Cleaved caspase-3 (*p=0.036). Values in parenthesis represent the level of statistical significance when compared with vehicle-treated controls. Results normalized to corresponding β-actin band densities.
Figure 2
Figure 2. Vorinostat persuaded apoptosis by influencing translocation of Bax protein to mitochondria
(A) Vorinostat treated and untreated A431 cells were stain with MitoTracker (Red) and Bax (green). Arrows in the representative photograph showed the translocation of Bax protein from cytosol to mitochondria in vorinostat (2 μM) treated A431 cells. (B) Western blot of Bax and Cleaved caspase-3 protein is significantly up-regulated in vorinostat (2 μM) treated A431 cell line. (C) Relative densitometry analysis of Bax (A431 *P=0.007) and Cleaved caspase-3 (A431 **P=0.001) in vorinostat (2 μM) treated cells.
Figure 3
Figure 3. Vorinostat reduces expression of various HDACs and enhances acetylated histone and non-histone proteins
(A) IHC analysis of HDAC1 and 2 in vehicle-treated control and vorinostat-treated tumors sections (40X magnification). (B) A431 cells were treated with vorinostat (2 μM) for 24 hrs and subjected to Immunofluorescence analysis (20X and 40X magnifications) with indicated antibodies. (C) WB analysis of HDACs (1, 2, 3, 6 and 7) of protein isolated from excised xenograft tumor tissue. (D) Relative densitometry analysis of HDAC1 (**p=0.004), HDAC2 (*p=0.025), HDAC3 (*p=0.016), HDAC7 (*p=0.032), HDAC6 (p= 0.768). (E&F) WB and relative densitometry analysis p53 (acetyl K386) (p=0.065), Acetyl-p53 (Lys379) (*p=0.020) and for Ac-Histone H3 (**p<0.0001).
Figure 4
Figure 4. Vorinostat blocks cell cycle progression and activation of ERK1/2
(A) WB analysis for cell cycle regulatory proteins in vehicle-treated control and vorinostat-treated group. Cyclin D1 (p=0.069), Cyclin D2 (p=0.220), Cyclin A (**p=0.002), Cyclin E (*p=0.015) and p21cip1/waf1 (*p=0.057). (B) Image J analysis of cell cycle regulatory proteins. Results normalized to corresponding β-actin band densities. (C) WB analysis of ERK1/2 signaling proteins: p-44/42 (p=0.379) and p-p44/42 (*p= 0.014). (D) Image J analysis for ERK1/2 proteins.
Figure 5
Figure 5. Vorinostat dampens cell survival signaling pathway
(A & B) WB and relative densitometry analysis for AKT/mTOR signaling markers. p-AKT1/2/3 Thr308 (*p=0.004), p-AKT1/2/3 ser473 (*p=0.001), AKT1/2/3 (*p=0.19) significantly reduced in vorinostat treatment group of mice. (C&D) WB and densitometry analysis of p-mTOR (*P=0.04) and total m-TOR (P=0.15) in vorinostat treated group represent significant reduction of p-mTOR level. (E&F) WB and densitometry analysis of p-mTOR (*P=0.027) and total mTOR (*P=0.009) levels in vorinostat (2μM) treated A431 cells. (G&H) WB and relative densitometry analysis mTOR signaling proteins, p-p70S6 kinase (**P=0.001), p70S6 kinase (P=0.976), p-S6 ribosomal protein (**P=0.004), S6 ribosomal protein (**P=0.005), p-4E-BP1 (**p=0.003) and 4E-BP1 (*p=0.033).
Figure 6
Figure 6. Vorinostat inhibits cell proliferation and colony formation in A431 cells
(A) MTT assay to check proliferation shown at 24 and 48 hrs following treatment with vorinostat at various concentrations in A431 cells. Data expressed as the percentage compared to untreated control cells. (B) Photograph for A431 cells treated with vorinostat (2 μM) for clonogenic/colony formation assay. (C) Graphical representation for colony formation assay. Results expressed as percentage survival relative to control ±SE (*p=0.021). (D) Over expression of myr-Flag-AKT in transduced A431 cells significantly restores the cell viability in vorinostat (1–5 μM) exposed cells at both 24 and 48 hrs treatment in comparison to normal A431 cells. (E) WB analysis of normal A431 and overexpressed myr-Flag-AKT transduced A431 cells. (F) Relative densitometry analysis showed Vorinostat (2 μM) significantly diminished the level of p-AKT1/2/3 thr 308 (*P=0.042) and p-AKT1/2/3 ser 473 (*P=0.006) and up-regulate the level of cleaved caspase-3 (**P=0.004) in normal A431 cells while myr-Flag-AKT over expressing cells do not show increased caspase 3 cleavage in the presence of vorinostat.
Figure 7
Figure 7. Vorinostat abrogates invasive tumor phenotype in A431 cells
(A) Photographs showing scratch/wound healing assay after treatment with or without vorinostat (0.5 μM). Photographs are shown at 0 and 24 hrs. (B) Cell migration was checked by Boyden chamber assay with (0.5 μM) of vorinostat (10Xmagnification). (C) Graph for migration assay. Results expressed as percentage of migratory cells compared to control (±SE) per microscopic field (*p=0.010).
Figure 8
Figure 8. Vorinostat enhances cell polarity and reduces mesenchyme character
(A) WB analysis for E-cadherin (*p=0.038), N-cadherin (*p=0.059) and fibronectin (p=0.074). (B) Image J analysis for EMT markers. (C) Immunofluorescence staining for EMT biomarkers in A431 cells following vorinostat treatment (2 μM) for 24 hrs (40 X magnifications).
Figure 9
Figure 9. Flow diagram depicting mechanism by which vorinostat blocked growth of cutaneous SCCs
Vorinostat reduces the expressions level of HDAC1&2. Additionally, HDAC3 and HDAC7 protein levels were also diminished in this study. This decrease in HDAC expression leads to enhanced acetylation of histone-H3 and non-histone proteins like p53. Vorinostat treatment reduced cell survival signaling pathways invoked by AKT phosphorylation at Ser473 and Thr308. Concomitant with this, a reduction in mTOR and ERK signaling pathways occur that together blocked growth of invasive cutaneous SCCs.
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