Background:
Hypopharyngeal carcinoma is characterized by high degree of malignancy. The most common
pathological type is squamous cell carcinoma (HSCC). Cisplatin (cis-diamminedichloroplatinum, CDDP) is one of the most
widely used chemotherapeutic drugs nowadays and cisplatin resistance is a major problem in current treatment strategies.
Clinical researches have reported that high autophagy level often caused insensitivity to chemotherapy, a common
phenomenon that greatly reduces therapeutic effect in cisplatin-resistant tumor cell lines. 3-methyladenine (3-MA), an
inhibitor of PI3K, plays a vital role in the formation and development of autophagosomes. Therefore, we speculate that the
use of 3-MA may reduce cisplatin resistance in hypopharyngeal squamous cell carcinoma (HSCC).
Methods:
Part I: Cisplatin-resistant FaDu cell line (Human hypopharyngeal squamous cell carcinoma cells) was established
and cultured. Cell counting kit-8 was used to detect drug resistance. Inverted microscope was used to observe the
morphological changes at different concentrations, then the survival rate was calculated. After MDC staining, the autophagic
vacuoles were observed by fluorescence microscopy. The expression of Beclin1 from each group was confirmed by RTPCR and Western blot method.
Part II: 3-MA was applied for cisplatin-resistant cells intervention, Beclin1 was knocked down by plasmid transfection. Cell
cycle was detected using flow cytometry assay, apoptosis with necrosis was detected by staining with propidium iodide
(PI). CCK-8 was used to observe the cell survival rate in each group. The expression of autophagy-related protein Beclin1,
LC3I, LC3II, Atg-5 and P62 in each group was verified by Western blot analysis.
Results:
Cisplatin-resistant FaDu cell line can be stably constructed by cisplatin intervention. Compared with normal group,
autophagy and its related protein Beclin1 expression was enhanced in cisplatin resistant FaDu cells. Autophagy inhibition
group showed significant cell cycle changes, mainly manifested by G1 arrest, increased apoptosis rate and significantly
decreased survival rate at 24h level. The number of autophagy vacuoles were significantly reduced in the 3-MA group.
Furthermore, Western blot showed that expression of Beclin1, lc3-I, lc3-II, atg-5 protein decreased significantly after 3-MA
intervention, while the expression of p62 up-regulated, which also confirmed autophagy flow was blocked.
Conclusion:
Our work confirmed that enhanced autophagy is an important cause of cisplatin resistance in FaDu cells. The
use of 3-MA can significantly reduce autophagy level and arresting its cell cycle, promote apoptosis and reverse the cisplatin resistance condition, this effect is partly mediated by inhibition of Beclin-1 expression. Our data provides a theoretical basis
for the application of 3-MA in overcoming cisplatin resistance in hypopharyngeal cancer.
Detection of Glutathione in Oral Squamous Cell Carcinoma Cells With a Fluorescent Probe During the Course of Oxidative Stress and Apoptosis