Honokiol induced cell apoptosis in human lung cancer cell lines (A549 cell). Honokiol also induces upregulation of Bax and Bak, downregulation of Bcl-xL, Bcl-2 and dysfunction of mitochondrial in lung cancer cells. Animal studies have revealed a dramatic reduction in tumor volume after 21 days of treatment. Besides, electroacupuncture zusanli acupoint (ST36) combined with intraperitoneal injection of Honokiol inhibits tumour growth in the mouse xenograft model of A549 cells. This study demonstrates that honokiol may be a novel anticancer agent for the treatment of lung cancer cells and electroacupuncture could amplify the chemo-therapeutic effect in lung cancer treatment.
Figures collection
Fig. 7 Honokiol induced the apoptosis of human lung cancer cells ( in MTT assay)
A549 cells were incubated with various concentrations of honokiol for 24 hr (A) or 48 hr (B), and the cell viability was examined by MTT assay. The IC50 values were examined about 20μM after 24 hr treatment. Results are expressed as the mean ± S.E. *, p < 0.05 compared with control.
Fig. 8 Honokiol induced the apoptosis of human lung cancer cells (in annexin V/PI double staining)
Cells were treated with vehicle or honokiol for 24 hr, the percentage of apoptotic cells was also analyzed by flow cytometric analysis of annexin V/PI double staining (A&B). Results are expressed as the mean ± S.E. *, p <
0.05 compared with control.
Fig. 9 Honokiol induced the apoptosis of human lung cancer cells (in TUNEL method)
Cells were treated with vehicle or various concentrations of Honokiol for 24 hr, the TUNEL positive cells were examined by flow cytometry. Results are expressed as the mean ± S.E. *, p < 0.05 compared with control.
Fig. 10 Honokiol induces apoptosis by triggering the mitochondrial apoptotic pathway.
A549 cells were incubated with Honokiol (20μM) for different time intervals, the Bax, Bak and Bcl-XL expressions were examined by Western blot analysis. Each experiment was repeated at least three times.
Fig. 11 Honokiol induces the activation of caspase-3 in A549 cells A549 cells were incubated with different concentrations of Honokiol (A).
Then we treated A549 cells in 20μM for different time intervals, and the caspase-3 activities were examined by caspase ELISA kit. On the other hand, the caspase-3 expressions were examined by Western blot analysis. Results are expressed as the mean ± S.E. *, p < 0.05 compared with control. Each experiment was repeated at least three times.
Fig. 12 Honokiol induced the implanted tumor of human lung cancer cells significantly reduced after 2 and 3 weeks
We established xenografts of A549 cells in nude mice; as tumors reached 100 mm3 in size, the mice were divided into four groups and treated with either vehicle, Honokiol or acupuncture. We measured and recorded the volume of the implanted tumor in dorsal side of mice once a week. We found that the volume of the implanted tumor was significantly reduced after 2 and 3 weeks in Honokiol group (HK) and electroacupuncture conbined with Honokiol group (EA+HK). Results are expressed as the mean ± S.E. *, p < 0.05 compared with control; #, p < 0.05 compared with honokiol-treated group.
Fig. 13 Honokiol induced the implanted tumor of human lung cancer cells significantly reduced in the end of experiment
BLI Procedure and Image Analysis showed the tumor size and bioluminescent intensity in the different groups. The volume of Honokiol group (HK) and electroacupuncture conbined with Honokiol group (EA+HK) was significantly reduced.
Fig. 14 the specimens in the different groups
In the end of experiment, we scarified the mice and the xenografts were removed. The specimens were weighted and processed for pathological analyses.We also found that the volume of the implanted tumor was significantly reduced in HK group and EA+HK group.
Fig. 15 The weights of the implanted tumor were significantly reduced in HK group and EA+HK group
In the end of experiment, we scarified the mice and the xenografts were removed. The specimens were weighted and processed for pathological analyses.We also found that the weights of the implanted tumor were significantly reduced in HK group and EA+HK group. Results are expressed as the mean ± S.E. *, p < 0.05 compared with control. Each experiment was repeated at least three times.
Fig. 16 Electroacupuncture and Honokiol may prevent body weight loss in the mouse xenograft model of A549 cells
We recorded body weight of every mouse once a week to inspect the other physical condition. In the control group, we noted that the body weight of the mice was lost inversely the volume of the implanted tumor enlarged at the same time. However, in the EA, HK, EA+HK groups, we found that the appearance of body weight loss was not occurred. Results are expressed as the mean ± S.E. *, p < 0.05 compared with control. Each experiment was repeated at least three times.
Fig. 17 Comparison 4 groups in the structural changes of A549 cells displayed by H&E stain ×400
Control and EA group : being regular in the size and appearance of A549 cells , darker in the staining
HK and EA+HK groups : being different in the arrangement of A549 cells , smaller in size , obvious in heteromorphism , and fewer in pyknosis
Fig. 18 Honokiol induces mitochondrial-dependent apoptosis in A549 cells xenograft model
ex vivo analysis of tumors excised from mice showed significantly increased Bax and Bak protein expression in the HK and HK+EA groups compared with that in the control group, as shown by decreasing the expression of Bcl-xL and Bcl-2. Each experiment was repeated at least three times.
Fig. 19 Honokiol induces apoptosis via caspase3 and cytochrome C mediated in A549 cells xenograft model
ex vivo analysis of tumors excised from mice showed significantly cytochrome C and caspase 3 protein expression in the HK and HK+EA groups also significantly exceeded the control group. Each experiment was repeated at least three times.
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致謝辭
感謝恩師 馮逸卿教授與許昇峰教授於就學期間悉心的指導和照 顧，並充實我的西醫骨科與中醫針灸的專業知識，引領我以積極、謹慎 的態度去面對浩瀚的科學領域，感謝您細心的修改和指正，使本篇論文 得以誕生，師恩浩蕩，難以言喻。
口試審查期間承蒙中山醫學大學醫學研究所 楊順發副教授對本篇 論文撥冗審查惠予寶貴意見，使本論文更加完善，在此獻上最真誠的謝 意。
另外需特別感謝實驗期間本校藥理學科 湯智昕教授提供實驗室的 所有資源和參與本實驗的設計和規劃，提供許多寶貴意見，並在實驗遭 遇困難時提供解決方法，也幫我爭取了 55 屆骨科醫學會和 24 屆生醫年 會口頭報告的機會。湯教授對我實驗生涯的諸多幫助和關懷，給我更寬
另外需特別感謝實驗期間本校藥理學科 湯智昕教授提供實驗室的 所有資源和參與本實驗的設計和規劃，提供許多寶貴意見，並在實驗遭 遇困難時提供解決方法，也幫我爭取了 55 屆骨科醫學會和 24 屆生醫年 會口頭報告的機會。湯教授對我實驗生涯的諸多幫助和關懷，給我更寬