Effects Of The Unfolded Protein Response Activation On Chemosensitivity Of Hypopharyngeal Cancer FaDu Cells In Response To Hypoxia

Head and neck squamous cell carcinoma (HNSCC) is one of the mostcommon malignant tumors, which is the third most common cancer indeveloping countries, and ranks6th around the world. Because of the insidiousonset, infiltrative growth, submucosal diffusion, higher rate of lymph nodemetastasis, the prognosis of HNSCC is poor. Up to now, the major therapy ofadvanced HNSCC has been still dependent mainly on operation combinedwith radiochemotherapy. Although improvement in the diagnosis andtreatment have been made in the past two to three decades, the overall5-yearsurvival rate remains almost unchanged. Resiatance to radiochemotherapywith local recurrence and metastasis is the leading cause of the poor prognosisin HNSCC. Therefore, it is crucial to revaluate the current understanding ofHNSCC, nveil the mechanisms of pathogenesis development and seekeffective new treatment strategies.A large number of studies have shown that, radiochemotherapeuticresistance was closely related with the tumor microenvironment hypoxia. Fastgrowth, increased oxygen consumption, vasculature disorders and insufficientblood supply are main features of solid tumors, all of which results in tumormicroenvironment hypoxia. Hypoxia contributes to cell cycle arrest, limiteddiffusion of drugs, and the changes in gene transcription activity, whichcontributes to tumor growth, angiogenesis, metastasis, thereby enhancing theradiochemotheraputic resistance. Studies have shown that tumormicroenvironment hypoxia is the key factor of poor prognosis in head andneck squamous cell carcinoma. Therefore, targeting the tumormicroenvironment hypoxia, will play a tremendous role in improving thetreatment of HNSCC. The severity of hypoxia in solid tumors is ranging from normoxic tocompletely anoxic, and different mechanisms of hypoxia tolerance dependeson different kinds of hypoxia. The latest research has shown that UPRdependent pathway is an important way of tumor hypoxia adaptation, which isparticularly activated in tumor tissues. Glucose-regulated protein78(GRP78)is a symbol factor and a key regulator of UPR, and plays an important role intumorigenesis and development of a variety of solid tumors. However,whether hypoxia can cause specific activation of UPR in HNSCC, and its rolein chemosensitivity of HNSCC is still unclear.In present study, the expression of UPR related protein GRP78andCHOP and its effects on the chemosensitivity in hypopharyngeal carcinomawas carried out. The purpose of the present study is to find effective ways toreduce hypoxia-related radioresistance of hypoxic treatment in HNSCC,aiming at providing new ideas and methodological basis for minimizing thechance of recurrence and impoving the prognosis. This study was divided intothree parts:Part one: Clinicopathological significance of UPR-related proteins GRP78and CHOP expression in hypopharyngeal carcinomaObjective: To investigate UPR-related proteins GRP78and CHOPexpression in hypopharyngeal carcinoma and the relationship between theirexpression and clinicopathologic features, and to explore their roles in thetumorigenesis and development of hypopharyngeal carcinoma.Methods:1The expression of GRP78and CHOP were detected byimmunohistochemical staining in47hypopharyngeal carcinoma specimensand12paracancerous normal hypopharyngeal mucosal tissues，and theirrelationships with clinicopathologic characteristics were evaluated.2The expression of GRP78and CHOP were detected by immunoblotanalysis in4hypopharyngeal carcinoma specimens and paracancerous normalhypopharyngeal mucosal tissues.Results: 1Clinical immunohistochemistry analysis.1.1The expression of GRP78and CHOP in hypopharyngeal carcinoma andparacancerous normal hypopharyngeal mucosal tissues. Results fromimmunohistochemistry showed that out of the47surgical specimens,41werepositive for GRP78(positive rate87.23%),26for CHOP (positive rate55.32%), while the positive rate of GRP78was33.33%and CHOP was0%in12paracancerous normal hypopharyngeal mucosal tissue, respectively.Compared with paracancerous normal hypopharyngeal mucosal tissues, thepositive expression of GRP78and CHOP in hypopharyngeal carcinoma weresignificantly increased (χ~2=12.512, P=0.000; χ~2=11.868, P=0.001).1.2The relationships between the expression of GRP78and CHOP andclinicopathologic characteristics. The positive rate of GRP78was94.87%inthe moderately to poorly differentiated tumors, and was significantly higherthan that in well differentiated tumors, the latter of which was only50%(χ~2=8.311, P=0.004). The positive rate of CHOP was58.97%in the moderateto poor differentiation cases, and37.5%in well differentiation cases, but thedifference was not statistically different (χ~2=0.522, P=0.470). The positive rateof GRP78was96.55%in stage T3-T4cases, which was significantly higherthan that in stage T1-T2cases being72.22%(χ~2=3.921, P=0.048). Thepositive rates of CHOP were51.72%and55.56%in patients with T3-T4tumors and T1-T2tumors, respectively, and there was no statisticallysignificant difference between the two (χ~2=0.065, P=0.798).The positive rateof GRP78was97.14%in the clinical stage Ⅲ-Ⅳ, which was significantlyhigher than that in clinical stage Ⅰ-Ⅱ, and in which the positive expressionrate was58.33%(χ~2=8.852, P=0.003). The positive rates of CHOP were57.14%and50%in clinical stage Ⅲ-Ⅳ and Ⅰ-Ⅱ, respectively, and there wasno statistically significant difference between them (χ~2=0.184, P=0.668).Compared with the cases without cervical lymph node metastasis, theexpression of GRP78was significantly increased in lymph node metastasiscases, and positive rate was100%(χ~2=7.499, P=0.006). Simultaneously, thepositive rate of CHOP was53.57%(χ~2=0.086, P=0.770). The expression of GRP78was correlated with histological grade, T staging, clinical staging andcervical lymph node metastasis, but not with age, gender and tumor location(P＞0.05). The expression of CHOP was not correlated with histological grade,T staging, clinical staging, cervical lymph node metastasis and otherclinicopathologic factors (all P＞0.05).2Western blotting showed that compared with paracancerous normalhypopharyngeal mucosal tissues, the expression of UPR related proteinsGRP78and CHOP were both significantly increased in hypopharyngealcarcinoma.Conclusions: The expression of UPR-related proteins GRP78and CHOPwere both significantly higher in hypopharyngeal carcinoma than that inparacancerous normal hypopharyngeal mucosal tissues, which indicates thatUPR is activated in hypopharyngeal carcinoma.Part two: Establishment stable transfection cell line with shRNA targetingGRP78in FaDu hypopharyngeal carcinoma cell by lentivirus infectionObjective: To establish a cell line stably transfected with shRNAtargeting GRP78in FaDu hypopharyngeal carcinoma cell by lentivirusinfection, and provide a cell model for further investigate the role of GRP78intumorigenesis and development of hypopharyngeal carcinoma.Methods:1Four shRNA fragments targeting GRP78gene sequence were designedand synthesized. RNAi lentivirus eukaryotic vectors were constructed byGeneCopoeia Company Limited, with the uninfected cells (blank) and cellsinfected with the shRNA lentivirus containing scrambled oligonucleotide(scshRNA) as control. The lentivirus vectors were transfected into293T cellsusing Lipofectamine2000, the obtained LV-GRP78-shRNA lentivirus wasinfected into FaDu cells, the expression of GRP78mRNA and protein wasdetected by Real Time-PCR and Western blot assay, to investigate the optimalGRP78-shRNA which had apparent knock-down capacity.2The optimal lentivirus vectors LV-GRP78-shRNA2and lentiviruspackaging plasmids were co-transfected into293T cells. FaDu cells were infected with lentivirus，and puromycin was used to select the stable GRP78interference hypopharyngeal carcinoma FaDu cells.3Fluorescence expression rate of stable GRP78interference cell linewas detected by flow cytometry.4The GRP78mRNA and protein level of FaDu cells were detected byreal-time PCR and Western blot assay.Results:1Lentivirus vectors LV-GRP78-shRNA2, LV-GRP78-shRNA3andLV-GRP78-shRNA4were transfected into293T cells, the expression ofGRP78mRNA and protein were both significantly decreased after transfection,among which the LV-GRP78-shRNA2knockdown effect was the mostsignificant.2Stable GRP78interference FaDu cell clones were selected by2.5μg/mlpuromycin.3Fluorescence expression rate in uninfected, scshRNA andGRP78-shRNA2FaDu cells were0.833%±0.115，99.93%±0.058and99.97%±0.058, respectively.4Real-time PCR assay showed that compared with scshRNA and blank,the expression of GRP78mRNA in LV-GRP78-shRNA2group wassignificantly decreased in stable GRP78interference FaDu cells (t=-24.900,P=0.000; t=-28.604, P=0.000), and there was no significant differencebetween the scshRNA and blank group (t=1.943；P=0.124).5Western blot assay showed that compared with scshRNA and blank,the expression of GRP78protein in LV-GRP78-shRNA2group wassignificantly decreased in stable GRP78silencing FaDu cells (t=-11.388,P=0.000; t=-13.004, P=0.000), and there was no significant differencebetween the scshRNA and blank group (t=0.066, P=0.951).Conclusions: Hypopharyngeal carcinoma FaDu cell line with GRP78gene stable interference by lentivirus vectors was successfully established,which provided a cell model for further study of the function of the gene. Part Three: Activation of UPR and the effects of GRP78-shRNA on thechemosensitivity under severe hypoxia conditions in hypopharyngealcarcinoma FaDu cellObjective: To observe the activation of UPR under different hypoxiaconditions, and explore the mechanism and effects of GRP78on thechemoresistance in response to hypoxia in hypopharyngeal carcinoma FaDucells.Methods:FaDu cells were cultured under normoxia and different hypoxic conditions,the expression of UPR related proteins were detected, the inhibition andapoptosis of cisplatin on FaDu cells were measured, and further explore theeffects of GRP78knockdown combined with cisplatin on proliferation andapoptosis related proteins.1The expression of UPR related proteins GRP78and CHOP weredetected at24h under different severity of hypoxia in hypopharyngealcarcinoma FaDu cell line by immunocytochemical method.2The expression of GRP78and CHOP protein were detected by Westernblotting in hypopharyngeal carcinoma FaDu cell line at the indicated periodsof times (3,6,9,12and24h) under different severity of hypoxia.3The inhibition of cisplatin on hypopharyngeal carcinoma FaDu cellsproliferation were detected by MTT assay under normoxic conditions (20%O2), moderate (1%O2) and severe hypoxia conditions (＜0.02%O2). Theeffects of GRP78knockdown combined with cisplatin on proliferation undernormoxic conditions and severe hypoxia conditions were further explored.4The effects of GRP78knockdown combined with cisplatin onapoptosis under normoxic and severe hypoxia conditions at24h weremeasured by the propidium iodide method using flow cytometry.5GRP78(+/+)and GRP78(-/-)FaDu cells were cultured for24h undernormoxic and severe hypoxia conditions and the expression of GRP78andCHOP were detected by immunocytochemical method.6The role of GRP78knockdown on regulation of the apoptosis-relatedproteins CHOP, Bcl-2and Bax were measured by immunoblot analysis in FaDu cells under normoxic and severe hypoxia conditions.Results:1The expression of UPR-related proteins GRP78and CHOP were detectedunder different severity of hypoxia in hypopharyngeal carcinoma FaDu cellline by immunocytochemical method: Immunocytochemical results showedthat compared with normoxic conditions，there were no significant changes inGRP78and CHOP expression in FaDu cells exposed to moderate hypoxia at24h (t=0.459, P=0.67; t=0.226, P=0.832). Compared with normoxicconditions，the expression of GRP78and CHOP were both significantlyincreased in severe hypoxia (<0.02%O2)(t=14.709, P=0.000; t=4.432,P=0.011). Compared with moderate hypoxia (1%O2), the expression ofGRP78and CHOP were both significantly increased in severe hypoxia(<0.02%O2)(t=13.055，P=0.000；t=3.85, P=0.018).2The expression of GRP78and CHOP protein were detected by Westernblotting in hypopharyngeal carcinoma FaDu cell line under different severityof hypoxia, the results showed that: compared with normoxic conditions, therewere no significant changes in GRP78and CHOP expression in FaDu cellsexposed to moderate hypoxia (1%O2) for the indicated period of time (3,6,9,12and24h)(P>0.05). HIF-1α, an important marker of hypoxia, was increasedgradually from6h to24h, and peaked at24h. Compared with normoxicconditions, the expression of GRP78protein was increased gradually from3hto9h under severe hypoxia(<0.02%O2), and persisted at high level from9h to24h. The difference was statistically significant (3h,6h, P<0.05;9h,12h,24h,P<0.01). The expression of CHOP protein was increased gradually from12hto24h, and peaked at24h (12h, P<0.05;24h, P<0.01). HIF-1α wassignificantly increased in3h severe hypoxia,6-12hours maintain a higherlevel, and observed an obviously decline at24h severe hypoxia, but stillremained higher than that in normoxic conditions. The difference wasstatistically significant (3h,6h,9h, P<0.01;12h,24h, P<0.05).3The results of MTT assay3.1The effects of cisplatin on the proliferation in FaDu cells uder different hypoxic conditions. MTT results showed that compared with normoxicconditions，the proliferation inhibition rates of cisplatin on FaDu cells weresignificantly decreased both under moderate and severe hypoxia conditions(F=25.692, P=0.013, P<0.05; F=181.063, P=0.000; P<0.01)，and the latterwas more significant. Compared with moderate hypoxia conditions, theproliferation inhibition rates of cisplatin on FaDu cells was significantlydecreased under severe hypoxia conditions (F=80.537, P=0.000; P<0.01).3.2The effects of GRP78knockdown combined with cisplatin on theproliferation in FaDu cells under normoxic and severe hypoxia conditions.MTT results showed that compared with normoxic conditions，the effects ofcisplatin on proliferation inhibition in GRP78(+/+)and GRP78(-/-)FaDu cellswere both significantly decreased under severe hypoxia conditions (F=84.153,P=0.000, P<0.01; F=211.988, P=0.000, P<0.01). The effects of cisplatin onproliferation inhibition in GRP78(-/-)cells were both significantly higher thanthat in GRP78(+/+)cells under normoxic and severe hypoxia conditions(F=75.301, P=0.000; P<0.01; F=138.500, P=0.000; P <0.01).4Apoptosis was measured by the propidium iodide method using flowcytometry. Compared with normoxic conditions, apoptosis of FaDu cells wassignificantly decreased under severe hypoxia conditions (t=-4.525, P=0.011,P<0.05), and compared with the apoptosis of GRP78(+/+)cells, there was nosignificant change under normoxic conditions in GRP78(-/-)cells (t=2.158,P=0.097, P>0.05), but was significantly increased under severe hypoxiaconditions (t=4.213, P=0.016, P<0.05). Apoptosis in GRP78(-/-)cells treatedwith cisplatin were significantly higher than that in GRP78(+/+)cells treatedwith cisplatin both under normoxic conditions and severe hypoxia conditions,and the latter is more significant (t=8.827, P=0.011, P<0.05; t=12.803,P=0.000, P <0.01).5The effects of GRP78knockdown on the expression of CHOP. Theimmunocytochemical results revealed that compared with the GRP78(+/+)cells,the expression of GRP78were significantly decreased both under normoxicand severe hypoxia conditions in GRP78(-/-)cells (t=-3.769, P=0.023, P<0.05; t=-6.742, P=0.003, P<0.01); the expression of CHOP was no significantchange under normoxic conditions (t=0.331, P=0.767, P＞0.05), butsignificantly increased under severe hypoxia, and the difference wasstatistically significant (t=5.616, P=0.005; P <0.01).6The effects of GRP78on the expression of apoptosis related-proteinswere measured by immunoblot analysis. Compared with the GRP78(+/+)cells,GRP78expression were significantly decreased in GRP78(-/-)cells both undernormoxic conditions and severe hypoxic conditions (t=-10.052, P=0.006,P<0.01; t=-12.209, P=0.006, P<0.01), and the protein level of CHOP wassignificantly increased under severe hypoxia for24h (t=3.418, P=0.025，P＜0.05). Compared with GRP78(+/+)cells, there was no significantly different ofCHOP under normoxic conditions in GRP78(-/-)cells (t=0.08, P=0.94; P＞0.05), but was significantly increased under severe hypoxic conditions (t=4.24,P=0.013; P<0.05). Compared with GRP78(+/+)cells, the expression of Bcl-2and Bax had no significant change in GRP78(-/-)cells under the normoxicconditions (P>0.05). Compared with normoxic conditions, Bcl-2wassignificantly increased under severe hypoxia (t=5.569, P=0.005; P<0.01), andBax was significantly decreased (t=-3.081, P=0.037; P<0.05) in GRP78(+/+)cells. The expression of Bcl-2was significantly decreased in GRP78(-/-)cells(t=-13.25, P=0.000; P<0.01) than that in GRP78(+/+)cells under severe hypoxia,and in contrast to Bcl-2, the expression of Bax was significantly increased inGRP78(-/-)cells (t=17.127, P=0.000; P <0.01).Conclusions:1UPR was activated by severe hypoxia in human hypopharyngealsquamous cell carcinoma FaDu cells, resulting in the increased expression ofGRP78and CHOP, but failed to activate by moderate hypoxia for theindicated period of time.2Hypoxia contributes to the chemoresisitance of hypopharyngealcarcinomacells, and the more severe hypoxia, the more significant resistanceto chemotherapy.3Induction of GRP78by severe hypoxia is associated with the proliferation and chemoresisitance of hypopharyngeal carcinoma cells.Knockdown of GRP78with shRNA can significantly inhibit cell proliferationability in FaDu cells and resulting in the increase of apoptosis, thechemosensitivity of FaDu cells was increased.4Knockdown of GRP78can significantly inhibit the expression ofBcl-2, and increase the expression of Bax and CHOP, thereby enhance thechemosensitivity under hypoxia conditions in hypopharyngeal carcinomaFaDu cell.5Hypopharyngeal carcinoma FaDu cells adapt to severe hypoxia mainlythrough UPR-dependent pathway. Down-regulation of UPR key regulatorGRP78may become a promising adjuvant treatment strategy for overcomingthe hypoxia tolerance and therapeutic resistance in HNSCC.