| Summary: | 博士 === 中山醫學大學 === 醫學分子毒理學研究所 === 97 === Part I
Our previous report indicated that HPV16/18 infection prevalence in female lung tumors was significantly higher than in male tumors. However, the different susceptibility to HPV infection between genders remains to be elucidated. IL-10 has been shown to suppress virus clearance to lead virus persistent infection in animal model, and it is conceivable that IL-10 is an important cytokine to regulate the homeostasis between TH1 and TH2 cytokine secretions. We therefore hypothesize that IL-10 mRNA expression level in female lung tumors may be higher than that in male tumors to explain why female has higher susceptibility to HPV infection. Herein, 121 lung tumors were enrolled to examine IL-10 mRNA and HPV DNA copy number by real-time PCR to verify whether IL-10 mRNA level is positively correlated with HPV infection and virus clearance. Our data showed that IL-10 mRNA levels in tumors from female and nonsmokers were higher than those in tumors from male and smokers (61.7% vs 37.7%, p = 0.008 for genders; 55.8% vs 34.3%, p = 0.032 for smoking status), and IL-10 mRNA levels were positively correlated with HPV infection in female tumors (p = 0.022), not in male tumors. Moreover, lung tumors with higher IL-10 mRNA levels had higher HPV DNA copy number than those with lower IL-10 mRNA levels (p = 0.030), suggesting that IL-10 expression may be associated with HPV persistent infection in lung tumors. To verify which molecule(s) could be involved in IL-10 transcriptional regulation, luciferase reporter and ChIP analysis showing that p53 may be an important transcription factor to upregulate IL-10 transcription. Additionally, 17-βestrodiol may synergistically enhance IL-10 transcriptional upregulation by p53 in p53 wild-type lung cancer cells. To elucidate whether lung cancer cells with higher IL-10 expression may be more suppression of TH1 cytokines expressions than those with lower IL-10 expression to modulate the the capability of virus clearance and tumor surveillance, two TH1 biomarkers, T-bet and IFN-γ were examined in PBMC from healthy donor which were cultured in the condition medium of TL-1 cells with higher IL-10 in comparison with those in the condition medium of TL-4 cells with lower IL-10 expression. Our data showed that T-bet and IFN-γ expressions in the condition medium of TL-1 cells were more suppressed than those in the condition medium of TL-4 cells. However, the suppression can be restored by the neutralization of IL-10 antiboy. The cytotoxicity of PBMC to TL-1 cells was significantly lower than that of PBMC to TL-4 cells. We also observed that patients with higher IL-10 mRNA plus HPV16/18 E6-positive expression had poorer prognosis than those with lower IL-10 mRNA plus HPV16/18 E6-negative expression. In summary, high expression of IL-10 may suppress the capability of virus clearance and tumor cell cytotoxicity to promote HPV-associated lung tumorigenesis.
Part II
The 5-year survival rate of non-small cell lung cancer remains around 15%, even though the improvement of the diagnostic techinology and new drug discovery are progress since two-decades. P53 is considered to play a key regulator on tumor progression, tumor recurrence, and chemo-therapeutic response. In the present study, 306 lung tumors and adjacent normal lung tissues were enrolled for p53 mutations and MDM2 309SNP by direct sequencing and PCR-RFLP, respectively. Among these, 266 adjacent normal lung tissues were collected to evaluate p53 codon 72 polymorphisms by PCR-RFLP, and 198 lung tumors were collected to determine MDM2 and GADD45 mRNA expressions by real-time PCR for assessment of p53 transcriptional activity. These data were used to verify whether p53 mutation, p53 codon72 genotypes, and MDM2 SNP309 genotypes and their combinations could be associated with tumor recurrence and prognosis. Our data indicated that patients harbored wild-type p53 combined with MDM2 GG genotype had the highest MDM2 and GADD45 mRNA expression levels among the four combinations, and stage-I patients harbored wild-type p53 combined with MDM2 GG genotype had more favorable prognosis than those with wild-type p53 combined with MDM2 TT genotype and p53 mutations. On the contrast, patients with p53 mutations had worser prognosis than those with wild-type p53. In cohort study, stage-I patients harbored wild-type p53 combined with MDM2 GG genotype had lower tumor recurrence rate compared with those harbored MDM2 TT/TG genotype or p53 mutation, but it did not reach statistically significance because a few case numbers were available (p = 0.087). On the other hand, patients with p53 codon 72 Pro allele had higher tumor recurrence rate than those with Arg/Arg genotype (p = 0.022). In addition, patients harbored p53 mutation combined with Pro allele had a higher trend in tumor recurrence rate compared with those harbored wild-type p53 combined with Arg/Arg genotype (p = 0.052), and a similar trend was also observed in stage-I patients (p = 0.052). In the prognostic significance of p53 codon 72 poymorphism, stage I patients harbored p53 mutation combined with Pro allele had poorer prognosis than those harbored wild-type p53 combined with Arg/Arg genotype (HR=2.66, 95% CI, 1.21-5.85, p = 0.02). Therefore, the information of p53 mutation, p53 codon 72 polymorphisms, and MDM2 SNP309 genotype obtained by simple methods may be feasible to assess the tumor recurrence and the clinical outcome of stage I lung cancer, and helpful to choose a favorable clinical strategy to improve patients’ outcome.
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