Clinical Significance of Circulating Serum Levels of sCD95 and TNF-α in Cytoprotection of Cervical Cancer

Abstract

Background: This study correlates the serum levels of sCD95 & TNF-α with a simple cell-based assay to evaluate the capacity of the serum sample to induce apoptosis in Jurkat cells. Interlinking of these parameters can be explored to design a minimum invasive diagnostic strategy for cervical cancer (CC).

Methods: Sera samples were assessed to induce apoptosis in Jurkat cells through FACS. Serum levels of sCD95 and TNF-α were measured by ELISA. JNK phosphorylation was evaluated in sera incubated Jurkat cells. Data was scrutinized through statistical analysis.

Results: Significantly higher serum levels of sCD95 and lower TNF-α levels were observed in CC patients; their sera samples inhibited induction of apoptosis in Jurkat cells through reduced JNK phosphorylation. Statistical analysis linked these three parameters for the early screening of CC.

Conclusion: Distinct sera levels of sCD95 & TNF-α in CC patients showed an anti-apoptotic effect, which can be considered for early detection of CC.

Keywords: Apoptosis; Jurkat Cells; Tumor Necrosis Factor-alpha; Uterine Cervical Neoplasms; sCD95.

Fig. 1

Normal sera induce apoptosis in the Jurkat cells as compared to the HEK-293 cells. Representative FACS dot-plot with 150 µl of healthy sera cocktail showing (A) Induction of apoptosis in Jurkat cells, whereas (B) HEK 293 cells did not show considerable induction of the apoptosis. (C) Jurkat cells and (D) HEK-293 cells, were incubated for 48 hrs with 0-200 µl cocktail of 20 healthy women sera. Apoptosis rate in each cell line was measured by flow cytometry using Annexin-V/PI markers. A total of 10000 events were captured by BD FACS Calibur flow cytometer system (USA) and analysis was performed with the CellQuesecPro software. The data are represented as means ±SEM from three independent experiments.

Fig. 2

Reduced JNK phosphorylation was observed during induced Apoptosis of Jurkat cells through the sera samples of CC patients. Jurkat cells were incubated with 100 µl serum of (A) Healthy women or (B) Cancerous women for 48 hrs. Cell were harvested after 48 hrs. followed by western blot and densitometry analysis to determine relative extent of JNK activation (ratio between integrated density of phospho-p54/46JNK and total JNK levels). Serum of healthy women induce the p54/46JNK phosphorylation as compared to cancerous patients’ sera.

Fig. 3

Differential parameters i.e. apoptosis induction in Jurkat cells as well as sCD95 & TNF-α concentration in sera samples of HC (n=20) Vs. CC patients (n=20). (A) Induction of apoptosis (%) in Jurkat cells after incubation with 100 µl sera samples of CC patients. (B) Increased levels of sCD95 (Fas receptor) in CC patients’ sera as compared to healthy sera samples. (C) Decreased levels of TNF-α in sera samples of CC patients Vs. HC. Concentrations of sCD95 and TNF-α were measured by ELISA in sera obtained from HC and CC patients separately in triplicate. These results are represented as mean ± Standard Error of mean. **p < 0.01 or ***p <0.001- as compared to HC.

Fig. 4

Correlation and best fit regression analysis for the serum concentration of sCD95 and TNF-α as well as apoptosis induction of Jurkat cells through sera samples of HC Vs. patients. Pearson correlation analysis was done to assess association between the variables. (A) Positive (direct) correlation (r=0.21) was observed between sCD95 and TNF-α concentration of sera in HC, whereas a negative (inverse) correlation (r=-0.22) was observed in CC patients. (B) Negative correlation was observed between sCD95 concentration and induced apoptosis for both HC (r=-0.07) and CC patients’ groups (r=-0.17). (C) For TNF-α concentration and induced apoptosis, HC showed a negative correlation (r=-0.04) in contrast to positive correlation (r=0.28) of CC patients.

Fig. 5

Receiver operative characteristics (ROC) curve analysis to check diagnostic accuracy for all the three variables and the cut off value to discriminate CC patients from HC. (A) Cut-off for Apoptosis induction (%) in Jurkat cells was found to be ≤ 10.33%, while its sensitivity and specificity is of 100.0%. (B) Cut-off concentration of sCD95 was found to be >1.9 ng/ml, while its sensitivity and specificity is of 95.0%. (C) Cut-off concentration of TNF-α was found to be ≤ 4.01 pg/ml, while its sensitivity and specificity is of 80.0%.