Hematoxylin, Millettia And Cisplatin Were Lung Cancer Cell Proliferation, Regulation Period

In recent years, malignant tumor has become a commonly-seen and frequently-occurring diseases, of which lung cancer has high morbidity. The death rate caused by lung cancer is increasing year by year. Due to the high morbidity and increasing death rate, more and more attention has been paid to treatment of tumors. At the same time, the effects Chinese medicine has in treating tumors have been recognized. Blood stasis syndrome is commonly found in the patients with malignant tumors. Whether the clinical symptoms or abnormal blood rheology, abnormal blood clotting and microcirculation obstacle, they are all closely related with tumors Hemorheologic agent are widely used in dealing with tumors. Thus, most researchers believe that they can inhibit the growth of tumors and contribute to defending the invasion and metastasis of tumors.Purpose:The research, based on previous clinical and experimental research, applies caesalpinia sappan, caulis spatholobi and combined cisplatin on mice planted Lewis lung carcinoma and discusses the inhibiting mechanism of caesalpinia sappan, caulis spatholobi and combined cisplatin on tumor growth by observing the inhibition of tumors, influences on cell cycles, change of protein and genetic expressions related with cell cycle and proliferation, hoping to provide a scientific basis for rational use of hemorheologic agent in the clinic treatment on tumors.Method:Inoculate the Lewis lung cancer tumors that grow well and are at log phase to the oxters of100mice and divide them into ten groups randomly, namely, blank group (K), cisplatin group (D), low dose caesalpinia sappan group (SL), high dose caesalpinia sappan group (SH), low dose caesalpinia sappan plus cisplatin group(SLD), high dose caesalpinia sappan plus cisplatin group (SHD), low dose caulis spatholobi group (JL), high dose caulis spatholobi group (JH), low dose caulis spatholobi group plus cisplatin(JLD), high dose caulis spatholobi plus cisplatin group (JHD). Give their gavage respective medicine and intraperitoneal injection24hours after inoculating and record the time of tumor formation. Put the mice to death after21days of gavage, take out their underarm tumor issues, weigh them and calculate the inhibiting rate according to the formula. After that, test out the cell cycle of primary tumor tissues by flow cytometry, examine the protein expression of PCNA and HIF-la by laser scanning confocal microscope and immunohistochemical technique, examine the protein expression of CyclinDl and CDK4by laser scanning confocal microscope, and at last test the genetic expression of RB1and CDKN2A.Results:(1) The forming time, weight and inhibition rate of primary tumor The forming time of tumors in caesalpinia sappan group, caulis spatholobi group, cisplatin group and the combined caesalpinia sappan and cisplatin group and caulis spatholobi and cisplatin group is relatively late compared with that of blank group. The tumor weights of drug groups are lower than those of the non drug groups. Except low dose caesalpinia sappan group (P>0.05), the rest groups all have statistical significance (P>0.05). The tumor weight of Chinese medicine plus cisplatin group is lower than that of cisplatin group, so high dose Chinese medicine plus sisplatin group has statistical significance compared with sisplatin group (P<0.05). The inhibition rate of the groups is ranked as follows:Chinese medicine plus chemotherapy group> cisplatin group> pure Chinese medicine group. The tumor inhibition rate of Chinese medicine plus chemotherapy group is higher than that of pure Chinese medicine group or pure chemotherapy group, which means Chinese medicine plus chemotherapy group is more effective in inhibiting tumors.(2) Change of primary tumor cell cycleBy adopting flow cytometry to test the respective groups’tumor in cell cycle, the research finds that except low dose caesalpinia sappan group and low dose caulis spatholobi group (P>0.05), the proportion of G0/G1period of cell circle in drug groups increases compared with non drug groups (P<0.05). Besides, the proportion of G0/G1period in the Chinese medicine plus cisplatin group increases compared with cisplatin group (P<0.05).(3) The protein expression of PCNA and HIF-la of primary tumorBy resorting to laser scanning confocal microscope and immunohistochemistry to test the expression of PCNA and HIF-la in primary tumor, the results show that the degree of PCNA expression in drug groups is lower than that of blank group, except low dose caesalpinia sappan group and low dose caulis spatholobi group (P>0.05), which has statistical significance (P<0.05). The PCNA expression in low dose Chinese medicine group plus cisplatin is lower than that in cisplatin group. However, the difference has no statistical significance (P>0.05). The PCNA expression in high dose Chinese medicine plus cisplatin group significantly decreased compared with cisplatin group (P<0.01). Except low dose caulis spatholobi group (P>0.05), the expressions of HIF-la in rest groups are lower than those of blank group (P<0.05). The expression HIF-la in Chinese medicine plus cisplatin group decreases compared with cisplatin group, but has no statistical significance (P>0.05).(4) Protein expressions of CyclinDl and CDK4of primary tumorAfter using laser scanning confocal microscope to examine protein expression of CyclinDl and CDK4of primary tumor, the results show that except low dose caesalpinia sappan group and low dose caulis spatholobi group (P>0.05), the CyclinD1expression in rest groups is lower than that of blank group (P<0.05), of which the CyclinD1expression in Chinese medicine plus cisplatin group is lower than that of cisplatin group, while the low dos caesalpinia sappan group plus cisplatin group has no statistical significance compared with cisplatin group (P>0.05). The CDK4expression in pure pure caesalpinia sappan and caulis spatholobi group has no significant difference from blank group (P>0.05). The CDK4expressions in cisplatin group and Chinese medicine plus cisplatin group decrease compared with that of the blank group (P<0.05). The CDK4expression in low dose Chinese medicine plus cisplatin group has no significant difference from cisplatin group (P>0.05). The CDK4expression in high dose Chinese medicine plus cisplatin group decreases compares with cisplatin group (P<0.05).(5) The genetic expressions of RB1and CDKN2A of primary tumorThrough adopting fluorescence quantitative PCR to test the genetic expressions of RB1and CDKN2A mRNA of primary tumor, the results show that the RBI gene mRNA expressions in drug groups are higher than those in blank group. However, the low dose caesalpinia sappan and caulis spatholobi group has no statistical significance compared with blank group (P>0.05). The RB1gene mRNA expression in Chinese medicine group plus cisplatin group increases compared with cisplatin group (P<0.05). The CDKN2A gene mRNA expressions in all groups increase compared with blank group, while pure caesalpinia sappan group and low dose caulis spatholobi group have no statistical significance compared with blank group (P>0.05). The CDKN2A gene mRNA expression in Chinese medicine plus cisplatin group increases compared with cisplatin group. However, only high dose caulis spatholobi plus cisplatin group has statistical significance compared with cisplatin groups(P<0.05).Conclusion:Caesalpinia sappan and caulis spatholobi and the combination of the caesalpinia sappan and cisplatin and that of caulis spatholobi and cisplatin are capable of inhibiting mice’s Lewis lung cancer growth and proliferation and the inhibition is also related with the dose of the drugs. Actually, the influences are mainly on cell cycles by impeding them in G0/G1period. Besides, it can also inhibit the PCNA ad HIF-la protein expression related with cell proliferation and adjust P16-CyclinDl-CDK4-RB route. Finally, the growth of tumor cells, as well as the production and growth of tumor are inhibited through the combined effects mentioned above.