Honokiol Suppresses Glioblastoma Cell Line Progression In Vitro And In Vivo

Background and ObjectiveGlioblastoma is the most common and lethal type of primary brain tumor, accounting for82%of cases of malignant glioma. Despite mμltimodal treatment including surgical resection, adjuvant TMZ-based chemotherapy and radio therapy, glioblastoma still remains invariably poor prognosis with only15months of the median overall survival time from diagnosis. It is now commonly recognized that rapid proliferation, the widely infiltrative growth and invariably aggressive biological behavior of glioblastoma cells lead to treatment failure and tumor recurrence. Therefore, the development of novel agents with promising therapeutic resμlts and minimized toxicities for the treatment of glioblastoma is urgently needed.In the past decades natural products have played a critical role in the drug discovery and development. Compared to the synthetic molecμles, natural products from plants, notably from traditional Chinese medicine (TCM), are more easily obtained and more cheap, with less toxicity. In addition, natural products from plants have been reported to exhibit antitumor acitivity by enhancing immunine function, inhibiting proliferation, suppressing angiogenesis, inducing apoptosis and differentiation, regμlating signaling pathways, inhibiting the telomerase activity, as well as their cytotoxicity to cancer cells. Therefore, the potential of natural products from plants has been recognized by the scientific community in the Western world and great efforts have been made to systematically investigate the active component of TCM for cancer therapy.Honokiol (3,5-di-(2-propenyl)-1,1-biphenyl-2,2-diol, HNK) is a natural bioactive molecμlar compound isolated from the Magnolia officinalis, a well-known Chinese traditional medicine Accumμlating studies demonstrated that honokiol exerts multiple pharmacological activities including anti-inflammatory, anti-bacterial, anti-angiogenic, anti-thrombocytic and anti-oxidative activity. Moreover, intense mechanistic and preclinical studies indicate that HNK exhibits potent antitumor activity against a variety of human cancer cell lines, including human multiple myeloma, colon cancer cell line RKO, leukemia cell lines HL-60and Molt4B, hepatocellμlar carcinoma SMMC-7721cells lung, pancreatic cancer cell lines MiaPaCa and PC-3, prostate cancer cells and so on, by inhibiting cell growth and inducing apoptosis or autophagy. Furthermore, HNK exhibited no appreciable toxicity. However, few studies have reported the antineoplastic effects of HNK on glioblastoma cells. Apoptosis induced by HNK and its associated pathway have yet to be discovered. In this study, we aim to investigate the therapeutic potential of HNK against glioblastoma cells in vitro, the biological properties of these tumor cells, including their morphology, proliferation, cell cycle, paraptosis, apoptosis, migration and invasion were studied. Moreover, to elucidate the mechanism of HNK treatment on glioblastoma, the key sinaling proteins in MAPK pathway and STAT3signaling pathway were examined. All the data provided the experimental support for the treatment of glioblastoma with HNK in clinic.Methods and ResultsPart1Effect of HNK on anticancer ability in glioblastomas cell lines.1.1Effect of HNK on proliferation of glioblastomas cell lines and cell cycle distribution.To determine the effect of HNK on the cellular proliferation in glioblastomas, CCK-8assay was performed, according to the protocol provided by the manufacturer. We performed dose-response and time course studies in three established cell lines including U251, U87, and T98G. Inhibition rate were calculated. Our resμlts showed that HNK markedly inhibited growth of all three cell lines in a dose-and time-dependent manner from5to40μg/ml HNK at12-72h. Based on the resμlts obtained in terms of cell growth inhibition, we found that U87cells and U251cells were more sensitive to HNK and then were selected for the following steps of our study.The cellular DNA content distribution of U87and U251cells were examined by flow cytometry and the mRNA expression of P21, p27, and cyclin E was determined by RT-PCR. The results showed that HNK induces a slight G0/G1arrest in U87and U251cells. Moreover, altered expression of cyclin E, p21and p27may account for an arrest in G0/G1phase. 1.2Effect of HNK on apoptosis of U87cells.Cell morphology was observeded under light microscopy after treatment with different concentration of HNK for24h. For nuclear morphology, cells were stained with Hoechst33342and were observed by a Nikon ECLIPSE Ti fluorescence microscope (Nikon, Japan) and photographed. It was noted that after HNK treatment, the number of U87cells decreased and a proportion of the cells shrank, detached from the culture plate and became round. Furthermore, membrane blebbing and formation of apoptotic bodies can be found when cells treated with higher concentration of HNK. Accordingly, Hoechst33342staining results showed that the nuclei of control cells were round and large in size, exhibiting homogeneous blue fluorescence. By contrast, parts of cells treated with10μg/ml HNK for24h exhibited condensed or fragmented nuclei which is the character of cell apoptosis. All of these findings suggested that HNK induced apoptosis of U87cells.To further confirm apoptosis-induced by HNK, Annexin V/PI double staining assay was performed by flow cytometry. A time-dependent study indicated that HNK at10μg/ml induced10.3%,25.1%, and26.4%of total apoptotic U87cells after12,24,48h treatment. While at20μg/mg, the ratio of apoptotic cells has no significant change, accompanied with elevated necrotic cells for8.5%,15.3%,45.4%.Mitochondrial transmembrane potential was monitored by flow cytometry using Rh123. Western blot was performed to detect the activities of effector caspase (caspase-3) and initiator caspase (caspase-8,-9), Bcl-2family proteins (Bcl-2,Bax),as well as Fas/FasL. The results showed that HNK-induced apoptosis is characterized by the activation of caspase-3,-8, and-9, loss of△Ψ, accompanied with elevated expression of Fas and FasL. In addition, HNK increased expression of Bax and decreased expression of Bcl-2, finally resulted in down-regulation of Bcl-2/Bax ratio.To verify whether caspases are necessary in apoptosis induced by HNK in U87cells, we pretreated cells with pan-caspase inhibitor Z-VAD-fmk prior to HNK exposure, then detected the cleaved caspase-3protein and the changes of apoptosis. We found though caspase-3activity was almost totally blocked by the pan-caspase inhibitor Z-VAD-FMK, the inhibitor does not block honokiol-induced apoptosis.We analyzed the activation of intracellμlar pathways related to glioblastomas development MAPK pathways and STAT3. Western blot was performed to detect the protein phosphorylation state of ERK, p38and JNK, STAT3in control cells and in cells treated for1,6,12h with HNK.There were no detectable changes in expression of total ERK, p38, JNK and phosphorylated-JNK protein level. By contrast, phosphorylation of ERK and STAT3were reduced with the time of HNK treatment within12h and obviously declined. Conversely, the treatment caused a significant elevation of the activation of p38..3Effect of HNK on paraptosis of U251cellsCell morphology was observeded under light microscopy after treatment with different concentration of HNK. For nuclear morphology, U251cells were stained with Hoechst33342and were observed by a Nikon ECLIPSE Ti fluorescence microscope (Nikon, Japan) and photographed. It was noted that HNK induced a dose-dependent cytoplasmic vacuolization in U251cells, fitted the criteria of paraptosis or autophagy. The trypan blue dye exclusion assay was perfomed to measure membrane integrity. Hoechst33342staining results showed that although HNK-induced nuclear condensation was observed, but without nuclear fragmentation, lack of apoptotic morphology.To exclude the possibility of apoptosis, Annexin V/PI double staning was performed by flow cytometry. The results exhibited no obvious apoptotic change in U251cells.To exclude the possibility of autophagy, marker of autophagy such as transformation of microtubule-associated protein1light chain3(LC3) Ⅰ to LC3Ⅱ was identified.Western blot results revealed that no significant LC3protein mordification after treatment with HNK.To further confirm the effect of HNK on U251cells, the ultra-structur of U251cells after treament with HNK was observed usinga Transmission Electron.The results revealed that HNK induced extensive cytoplasmic vacuolization in U251cells, swelling of endoplasmic reticulum (ER) and mitochondria.In addition, pretreatment with pan caspase inhibitor can’t block the cytoplasmic vacuolization and the cell growth inhibition induced by HNK.We analyzed the activation of intracellμlar pathways related to glioblastomas development MAPK pathways and STAT3. Western blot was performed to detect the protein phosphorylation state of ERK, p38and JNK, STAT3in control cells and in cells treated for1,6,12h with HNK. there were no detectable changes in expression of total ERK, p38, JNK protein level. By contrast, phosphorylation of STAT3were reduced with the time of HNK treatment within12h and obviously declined. Conversely, the treatment caused a significant elevation of the activation of p38,ERK and JNK.1.4Effect of HNK on migration of and invasion of glioblastoma cells U87and U251.The wound healing assay and Transwell invasion assay were used to assess the effects of HNK on the migration and invasion capacity of U87and U251cells. The sites of the wound line were photographed immediately after scratching and24h later. The width of the wound line was measured, and the migration distances were tocalculated relative to the control group. According to our data, HNK modestly inhibited the motility of U87and U251cells at5ug/ml, and more obviously at lOug/ml. The same results was obtained in Transwell invasion assay.U87and U251cells were serum starved for6h, and treated with various concentration of HNK.24h later, the effect of HNK on MMP-2and MMP-9expression was measured by Western blot. The results showed that the down-regulation of MMP-2and MMP-9were both contributed to migration of and invasion of glioblastoma cells U87and U251.Part2HNK inhibited GBM growth in vivoTo evaluate whether HNK inhibits GBM progression in vivo, human glioblastoma cells U251were injected into the right flank of nude mice. Treatment were started on7days after U251cells injections. HNK and PBS were administered intraperitoneally for14days. Tumor volumes were calculated and the weight of nude rice was detected. Survivin was investigated by ICH. Compared with control group, the weight did not change significantly after treated by HNK(p>0.05); while xenograft tumor growth was significantly slower than the control group after administration; tumor volume was significantly less in HNK treated group than the control group (p<0.05). In addition, The expression of survivin protein in HNK group decreased significantly.ConclusionsIn conclusion, the present study confirmed that HNK inhibited proliferation of glioblastoma cells by causing a slight G0/G1phase cell cycle arrest and inducing apoptosis or paraptosis. Here we demonstrated for the first time that HNK triggered apoptosis of glioblastoma cell line U87through both caspase-independent and caspase-dependent pathways, the latter was initiated with the extrinsic pathway and intrinsic pathway. Moreover, the inhibition of STAT3signaling, ERK1/2as well as activation of p38MAPK signaling pathway may be involed in apoptosis induced by HNK in U87cells.HNK induced paraptosis of U251cells, which were mediated by MAPK signaling and STAT3signaling.HNK also inhibited the cell migration and invasion of glioblastomas cells by down-regulation of MMP-2and MMP-9. In addition, HNK exhibited anticancer ability in glioblastomas cells in vivo. Our findings suggest that HNK treatment could be a promising therapeutic strategy in human glioblastoma.