| Head neck squamous cell carcinoma (HNSCC) occupied the sixth top incidence ofmalignancies worldwide. Patients suffered from the disease are always diagnosed asadvanced malignancy due to obscure symptom of early HNSCC. To date, the therapy ofadvanced HNSCC has been still dependent on surgery combined with radiochemotherapy. Inaddition to deprivation of normal speech and swallow by therapy, however, the survivalhasn't been improved the last decades. Therefore, better understanding the potential causesunderlying therapeutic failure is necessary to seek effective anti-cancer strategies.There exist poorly oxygenated lesions in most solid tumors due to either rapidproliferation as well as undue consumption of oxygen by tumor cells or aberrant structure ofvessels. Increasing data reported that hypoxia is associated with poor prognosis of most solidtumor including HNSCC. The direct cause is that it is difficult for anti-cancer agents todeliver into hypoxic lesions. In addition, hypoxic tumor cells activate many responsiblefactors to adapt hypoxia and even confer therapy resistance. Among these factors, hypoxiainducible factor1α (HIF-1α) displays a critical role since HIF-1α acts as a central core of thehypoxia-associated complex network. HIF-1α consists of an oxygen-dependent α subunitand a constant expressed β subunit. Under normoxia, α subunit is rapidly degraded though anubiquitin-proteasome pathway and keeps stable under hypoxia. Phosphorylated HIF-1α istransferred into nucleus and binds to hypoxia response elements (HREs) of gene totranscribe series of the downstream of HIF-1α including anti-apoptosis factors, such as Bcl-2,Survivin and XIAP, multiple drug resistance (MDR) gene and its product, P-glycoprotein(P-gp), and VEGF and other more than100factors mediating metabolisms and PH.However, hypoxia is an ambiguous concept because the distance between tumor cellsand vessels can influence the effects of hypoxia on genotype and phenotypes of tumor cells.For tumor cells that locate far from vessels, there is a diffusion-limited delivery of oxygen tothem resulting to a relatively stable and continuing oxygen pressure, called chronic hypoxia.However, cells close to vessels are always subjecting to repeated hypoxia/reoxygenation,named as cyclic hypoxia, intermittent hypoxia or fluctuating hypoxia. The former has been studied comprehensively and usually serve as a common hypoxic condition of most labs,however, to the latter, a recent recognition, the potential mechanisms by which cyclichypoxia induce malignant action of tumor cells remain to be determined. Currently, the fewefforts played on the investigation demonstrated that cyclic hypoxia displays more potentialrole on tumor therapeutic resistance and metastasis. It has been demonstrated by manystudies that increased expression of HIF-1α contributes to cyclic hypoxia-induced resistance.Moreover, to our knowledge, cycling hypoxia induced chemosensitivity and relatedmechanisms have been studied rarely, especially blank in HNSCC.Cancer stem cells (CSCs) are a heterogeneous subgroup of tumor bulk. Like stem cellsin normal tissue, CSCs have been believed to be the origination of tumor and they canself-renew rather than differentiate to programmed death as other cells. Intriguingly, theheterogeneous subset exhibits a powerful resistance to therapy. Therefore, therapeuticfailures are attributed to residual CSCs following with therapy, although tumor bulksignificantly shrink or even disappear. Current opinions think that CSCs are associated withhypoxic microenvironment closely. A hypothesis by us previously proposing that CSCs maybe mostly maintained by cycling hypoxia has been published in journal of medicalhypotheses. This present study provided evidence to the hypothesis.Objective:(1) To study acute cyclic hypoxia induced chemoresistance in laryngealcarcinoma cells as well as underlying mechanisms.(2) To investigate chronic cyclic hypoxiainduced chemoresistance in laryngeal carcinoma cells.Method:(1) To detect the sensitivity of Hep-2cells to DDP under different hypoxia byMTT assay; cellular cycle distribution was studied by flow cytometry and cycle associatedproteins, P27, Cyclin D1and CDK4were analyzed by western blot; To study STAT3,p-STAT3and HIF-1α expressions under any group by western blot; To determine the role ofHIF-1α under cyclic hypoxia, Hep-2cell line with stable transfection of HIF-1α shRNA wasestablished;(2) the ratio of CD133+Hep-2cells was analysed through flow cytometry;(3)Hep-2cells were exposed to repeated cycles of hypoxia/reoxygenation to establish chroniccyclic hypoxia conditioned Hep-2cells (CIH hep-2cells); to detect chemosensitivity of bothHep-2and CIH hep-2cells by MTT; to study factors with proliferation as well as badprognosis by western blot.Result:(1)MTT assay showed that acute cyclic hypoxia can induce a more significant resistance to both long-term and short-term DDP treatment induced cytotoxicity comparedwith chronic hypoxia and normoxia; the results of flow cytometry showed that cycledistribution was not the cause of cyclic hypoxia induced chemoresistance; western blotshowed that HIF-1a presented overexpressed in a cycle of hypoxia/reoxygenation dependentmanner and kept stable during reoxygenation; silencing HIF-1a could reverse cyclic hypoxiainduced chemoresistance; constitutive active STAT3regulated cyclic hypoxia assoctiatedelevation of HIF-1α; Acute cyclic hypoxia can enrich CD133+cells in Hep-2cells.(2)Established CIH Hep-2cells had a higher capacity of proliferation as well as significantresistance to DDP; western blot showed that CIH Hep-2cells overexpressed VEGF andp-STAT3.Conclusion:(1) Our results described in the present study suggested that acute cyclichypoxia-induced chemoresistance in HNSCC depended on high expression of HIF-1α ratherthan cycle arrest.(2) Constitutive activation of STAT3regulated cyclic hypoxia associated expression ofHIF-1α.(3)The chemoresistance was not the cause of selection pressure(4) Acute cyclic hypoxia enriched CD133+CSCs(5) Chronic cyclic hypoxia-induced resistance may result from intrinsic multifacterouschanges.
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