Date of Award

2021

Degree Name

Pharmaceutical Sciences

College

School of Pharmacy

Type of Degree

M.S.

Document Type

Thesis

First Advisor

Dr. A.R.M.Ruhul Amin, Committee Chairperson

Second Advisor

Dr. Piyali Dasgupta

Third Advisor

Dr. Jeremy McAleer

Abstract

Upper aerodigestive tract cancers including cancers of the oral cavity, pharynx, larynx, esophagus, and lungs are the most prevalent cancers and leading causes of cancer-related deaths. Collectively, over 300,000 new cases and 146,500 deaths are projected within the US in the year 2021. Drug-associated toxicities, as well as resistance to therapy (intrinsic and acquired), are big challenges for successfully treating these cancers. Recent studies have shown that combining low-dose actinomycin D with existing therapies is a promising strategy to reduce toxicity (cyclotherapy) and to overcome resistance. The development of these treatment strategies however requires an understanding of the molecular mechanisms of the antitumor activity of actinomycin D as well as that of drug resistance. This study evaluated the mechanism of actinomycin D-induced apoptosis and its effects on p53 signaling pathway in aerodigestive tract cancers using in vitro models of lung and head and neck cancer. We determined the IC50 of actinomycin D in a range of aerodigestive tract cancer cell lines using the SRB assay at 72 h which spanned between 0.02nM -2.96nM. Subsequently, we measured apoptosis by using the Annexin V-PE staining. FlowJo was used to quantify apoptosis. Cleavage of PARP and caspase 3 was used to confirm apoptosis. We found that actinomycin D induced apoptosis in all cell lines tested, but the sensitivity varies between cell lines. Mechanistic studies revealed that actinomycin D time- and dose-dependently increased the expression of p53 and its downstream targets p21 and PUMA in cells with wild-type p53. Consequently, we explored the role of p53 in actinomycin D-induced apoptosis by ablating the expression of p53 using p53 shRNA. Interestingly, the ablation of p53 decreased the expression of p21 and PUMA in A549 cell line and p21 in H460 cell line. More importantly, ablation of p53 significantly protected cells from actinomycin D-induced apoptosis. Furthermore, we explored the mechanism of apoptosis in cell lines lacking wild type p53 and focused on the expression of p73, p21, and PUMA. Through the completion of these experiments, we can conclude that actinomycin D exerts apoptosis via p53-dependent and –independent mechanisms. These results will guide future combinatorial studies involving actinomycin D.

Subject(s)

Drug targeting -- Research.

Neck -- Cancer -- Research.

Dactinomycin -- Research.

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