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Non-small-cell lung cancer (NSCLC) is the most common type of lung cancer worldwide, and treatment outcomes are still poor. Magnolol, a hydroxylated biphenyl isolated from Magnolia officinalis, was found to be effective against hepatocellular carcinoma via inactivating nuclear-factor-kappa B (NF-B) signaling. However, whether magnolol targets not only NF-B but also other factors in NSCLC and may contribute to the suppression of tumor progression is unclear.Cell viability, flow cytometry, and western blotting assays were used to identify the mechanism of magnolol action in human lung adenocarcinoma cell lines A549 and CL1-5-F4.Our results indicated that magnolol induced cytotoxicity through extrinsic/intrinsic apoptosis signaling and suppressed phosphorylation of signal transducer and activator of transcription 3 (STAT3)/NF-B and expression of their downstream proteins.Magnolol not only induced extrinsic and intrinsic apoptosis signaling but also inactivated STAT3/NF-B and attenuated their signaling of epithelial-mesenchymal transition and metastasis-related protein expression in NSCLC.

Background/aim Ally lisothiocyanate (AITC), a constituent of naturally occurring isothiocyanates (ITCs) found in some Brassica vegetables, has been previously demonstrated to have anti-carcinogenic activity. However, there is no available information showing that AITC induces DNA damage and alters DNA damage repair proteins in human breast cancer MCF-7 cells. Materials and methods In the present study, we investigated the effects of AITC on DNA damage and repair responses in human breast cancer MCF-7 cells in vitro. Cell viability was measured by flow cytometric assay. DNA condensation (apoptotic cell death) and DNA fragmentation (laddered DNA) were assayed by DAPI staining and DNA gel electrophoresis assays, respectively. Furthermore, DNA damage (comet tail) was measured by the comet assay. Western blotting was used to measure the expression of DNA damage- and repair-associated proteins. Results AITC decreased cell viability in a dose-dependent and induced apoptotic cell death (DNA condensation and fragmentation) and DNA damage in MCF-7 cells. AITC increased p-ATMSer1981, p-ATRSer428, p53, p-p53Ser15, p-H2A.XSer139, BRCA1, and PARP at 10-30 μM at 24 and 48 h treatments. However, AITC decreased DNA-PK at 24 and 48 h treatment, and decreased MGMT at 48 h in MCF-7 cells. Conclusion AITC induced cytotoxic effects (decreased viable cell number) through induction of DNA damage and condensation and altered DNA damage and repair associated proteins in MCF-7 cells in vitro.

The metastasis of human osteosarcoma (OS) shows a difficult‑to‑treat clinical scenario and results in decreased quality of life and diminished survival rates. Finding or developing novel treatments to improve the life quality of patients is urgent. Bisdemethoxycurcumin (BDMC), a natural product, was obtained from the rhizome of turmeric (

Background/aim Demethoxycurcumin (DMC), one of the derivatives of curcumin, has been shown to induce apoptotic cell death in many human cancer cell lines. However, there is no available information on whether DMC inhibits metastatic activity in human glioblastoma cancer cells in vitro. Materials and methods DMC at 1.0-3.0 μM significantly decreased the proliferation of GBM 8401 cells; thus, we used 2.0 μM for further investigation regarding anti-metastatic activity in human glioblastoma GBM 8401 cells. Results The internalized amount of DMC has reached the highest level in GBM 8401 cells after 3 h treatment. Wound healing assay was used to determine cell mobility and results indicated that DMC suppressed cell movement of GBM 8401 cells. The transwell chamber assays were used for measuring cell migration and invasion and results indicated that DMC suppressed cell migration and invasion in GBM 8401 cells. Gelatin zymography assay was used to examine gelatinolytic activity (MMP-2) in conditioned media of GBM 8401 cells treated by DMC and results demonstrated that DMC significantly reduced MMP-2 activity. Western blotting showed that DMC reduced the levels of p-EGFR(Tyr1068), GRB2, Sos1, p-Raf, MEK, p-ERK1/2, PI3K, p-Akt/PKBα(Thr308), p-PDK1, NF-κB, TIMP-1, MMP-9, MMP-2, GSK3α/β, β-catenin, N-cadherin, and vimentin, but it elevated Ras and E-cadherin at 24 h treatment. Conclusion DMC inhibited cancer cell migration and invasion through inhibition of PI3K/Akt and NF-κB signaling pathways in GBM 8401 cells. We suggest that DMC may be used as a novel anti-metastasis agent for the treatment of human glioblastoma cancer in the future.

Some clinically used anti-cancer drugs are obtained from natural products. Allyl isothiocyanate (AITC), a plant-derived compound abundant in cruciferous vegetables, has been shown to possess an anti-cancer ability in human cancer cell lines in vitro, including human brain glioma cells. However, the anti-cancer effects of AITC in human glioblastoma (GBM) cells in vivo have not yet been examined. In the present study, we used GBM8401/

Background/Aim: Ouabain, isolated from natural plants, exhibits anticancer activities; however, no report has presented its mechanism of DNA damage induction in human osteosarcoma cancer cells in vitro. The aim of this study was to investigate whether ouabain induces DNA damage and repair, accompanied with molecular pathways in human osteosarcoma cancer U-2 OS cells in vitro. Materials and Methods: The percentage of viable cell number was measured by flow cytometric assay; DNA damage was assayed by DAPI staining, comet assay, and agarose gel electrophoresis. DNA damage and repair associated protein expressions were assayed by western blotting assays. Results: Ouabain reduced total cell viability, induced chromatin condensation, DNA fragmentation, and DNA damage in U-2 OS cells. Ouabain increased p-ATM(Ser1981), p-ATR(Ser428), and p53 at 2.5-10 μM, increased p-p53(Ser15) at 10 μM; however, it decreased p-MDM2(Ser166) at 2.5-10 μM. Ouabain increased p-H2A.X(Ser139), MDC-1, and PARP at 2.5-10 μM and BRCA1 at 5-10 μM; however, it decreased DNA-PK and MGMT at 2.5-10 μM in U-2 OS cells at 48 h treatment. Ouabain promoted expression and nuclear translocation of p-H2A.X(Ser139) in U-2 OS cells and this was confirmed by confocal laser microscopy. Conclusion: Ouabain reduced total viable cell number through triggering DNA damage and altering the protein expression of DNA damage and repair system in U-2 OS cells in vitro.

Background Tetrandrine, a bis-benzylisoquinoline alkaloid, induces apoptosis of many types of human cancer cell. Hydrogen peroxide (H2O2) is a reactive oxygen species inducer; however, there are no reports to show whether pre-treatment of tetrandrine with H2O2 induces more cell apoptosis than H2O2 alone. Thus, the present study investigated the effects of tetrandrine on H2O2-induced cell apoptosis of human keratinocytes, HaCaT, in vitro. Materials and methods HaCaT cells were pre-treated with and without tetrandrine for 1 h, and then treated with H2O2 for examining cell morphological changes and cell viability using contrast-phase microscopy and propidium iodide (PI) exclusion assay, respectively. Cells were measured apoptotic cell death by using annexin V/PI double staining and further analyzed by flow cytometer. Cells were further assessed for DNA condensation using 2-(4-amidinophenyl)-6-indolecarbamidine staining. Western blotting was used to measure expression of apoptosis-associated proteins and confocal laser microscopy was used to measure the protein expression and nuclear translocation from the cytoplasm to nuclei. Results Pre-treatment of tetrandrine for 1 h and treatment with H2O2 enhanced H2O2-induced cell morphological changes and reduced cell viability, whilst increasing apoptotic cell death and DNA condensation. Furthermore, tetrandrine significantly increased expression of reactive oxygen species-associated proteins such as superoxide dismutase (Cu/Zn) and superoxide dismutase (Mn) but significantly reduced the level of catalase, which was also confirmed by confocal laser microscopy. It also increased expression of DNA repair-associated proteins ataxia telangiectasia mutated, ataxia-telangectasia and Rad3-related, phospho-P53, P53 and phosphorylated histone H2AX, and of pro-apoptotic proteins BCL2 apoptosis regulator-associated X-protein, caspase-3, caspase-8, caspase-9 and poly ADP ribose polymerase in HaCaT cells. Conclusion These are the first and novel findings showing tetrandrine enhances H2O2-induced apoptotic cell death of HaCaT cells and may provide a potent approach for the treatment of proliferated malignant keratinocytes.

Leukemia is one of the major diseases causing cancer-related deaths in the young population, and its cure rate is unsatisfying with side effects on patients. Fluorouracil (5-FU) is currently used as an anticancer drug for leukemia patients. Casticin, a natural polymethoxyflavone, exerts anticancer activity against many human cancer cell lines in vitro, but no other reports show 5-FU combined with casticin increased the mouse leukemia cell apoptosis in vitro. Herein, the antileukemia activity of 5-FU combined with casticin in WEHI-3 mouse leukemia cells was investigated in vitro. Treatment of two-drug combination had a higher decrease in cell viability and a higher increase in apoptotic cell death, the level of DNA condensation, and the length of comet tail than that of 5-FU or casticin treatment alone in WEHI-3 cells. In addition, the two-drug combination has a greater production rate of reactive oxygen species but a lower level of Ca2+ release and mitochondrial membrane potential (ΔΨm ) than that of 5-FU alone. Combined drugs also induced higher caspase-3 and caspase-8 activities than that of casticin alone and higher caspase-9 activity than that of 5-FU or casticin alone at 48 hours treatment. Furthermore, 5-FU combined with casticin has a higher expression of Cu/Zn superoxide dismutase (SOD [Cu/Zn]) and lower catalase than that of 5-FU or casticin treatment alone. The combined treatment has higher levels of Bax, Endo G, and cytochrome C of proapoptotic proteins than that of casticin alone and induced lower levels of B-cell lymphoma 2 (BCL-2) and BCL-X of antiapoptotic proteins than that of 5-FU or casticin only. Furthermore, the combined treatment had a higher expression of cleaved poly (ADP-ribose) polymerase (PARP) than that of casticin only. Based on these findings, we may suggest that 5-FU combined with casticin treatment increased apoptotic cell death in WEHI-3 mouse leukemia cells that may undergo mitochondria and caspases signaling pathways in vitro.

Bisdemethoxycurcumin (BDMC) has biological activities, including anticancer effects in vitro; however, its anticancer effects in human glioblastoma (GBM) cells have not been examined yet. This study aimed to evaluate the tumor inhibitory effect and molecular mechanism of BDMC on human GBM 8401/luc2 cells in vitro and in vivo. In vitro studies have shown that BDMC significantly reduced cell viability and induced cell apoptosis in GBM 8401/luc2 cells. Furthermore, BDMC induced apoptosis via inhibited Bcl-2 (anti-apoptotic protein) and increased Bax (pro-apoptotic proteins) and cytochrome c release in GBM 8401/luc2 cells in vitro. Then, twelve BALB/c-nude mice were xenografted with human glioblastoma GBM 8401/luc2 cancer cells subcutaneously, and the xenograft nude mice were treated without and with BDMC (30 and 60 mg/kg of BDMC treatment) every 3 days. GBM 8401/luc2 cell xenografts experiment showed that the growth of the tumors was significantly suppressed by BDMC administration at both doses based on the reduction of tumor size and weights. BDMC did not change the body weight and the H&E histopathology analysis of liver samples, indicating that BDMC did not induce systemic toxicity. Meanwhile, treatment with BDMC up-regulated the expressions of BAX and cleaved caspase-3, while it down-regulated the protein expressions of Bcl-2 and XIAP in the tumor tissues compared with the control group. This study has demonstrated that BDMC presents potent anticancer activity on the human glioblastoma GBM 8401/luc2 cell xenograft model by inducing apoptosis and inhibiting tumor cell proliferation and shows the potential for further development to the anti-GBM cancer drug.

Phenethyl isothiocyanate (PEITC), extracted from cruciferous vegetables, showed anticancer activity in many human cancer cells. Our previous studies disclosed the anticancer activity of PEITC in human glioblastoma multiforme (GBM) 8401 cells, including suppressing the cell proliferation, inducing apoptotic cell death, and suppressing cell migration and invasion. Furthermore, PEITC also inhibited the growth of xenograft tumors of human glioblastoma cells. We are the first to investigate PEITC effects on the receptor tyrosine kinase (RTK) signaling pathway and the effects of proinflammatory cytokines on glioblastoma. The cell viability was analyzed by flow cytometric assay. The protein levels and mRNA expressions of cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), were determined by enzyme-linked immunosorbent assay (ELISA) reader and real-time polymerase chain reaction (PCR) analysis, respectively. Furthermore, nuclear factor-kappa B- (NF-κB-) associated proteins were evaluated by western blotting. NF-κB expression and nuclear translocation were confirmed by confocal laser microscopy. NF-κB binding to the DNA was examined by electrophoretic mobility shift assay (EMSA). Our results indicated that PEITC decreased the cell viability and inhibited the protein levels and expressions of IL-1β, IL-6, and TNF-α genes at the transcriptional level in GBM 8401 cells. PEITC inhibited the binding of NF-κB on promoter site of DNA in GBM 8401 cells. PEITC also altered the protein expressions of protein kinase B (Akt), extracellular signal-regulated kinase (ERK), and NF-κB signaling pathways. The inflammatory responses in human glioblastoma cells may be suppressed by PEITC through the phosphoinositide 3-kinase (PI3K)/Akt/NF-κB signaling pathway. Thus, PEITC may have the potential to be an anti-inflammatory agent for human glioblastoma in the future.

Phenethyl isothiocyanate (PEITC), extracted from cruciferous vegetables, showed anticancer activity in many human cancer cells. Our previous studies disclosed the anticancer activity of PEITC in human glioblastoma multiforme (GBM) 8401 cells, including suppressing the cell proliferation, inducing apoptotic cell death, and suppressing cell migration and invasion. Furthermore, PEITC also inhibited the growth of xenograft tumors of human glioblastoma cells. We are the first to investigate PEITC effects on the receptor tyrosine kinase (RTK) signaling pathway and the effects of proinflammatory cytokines on glioblastoma. The cell viability was analyzed by flow cytometric assay. The protein levels and mRNA expressions of cytokines, including tumor necrosis factor

Background/aim Demethoxycurcumin (DMC), a derivate of curcumin from natural plants, exerts antitumor effects on various human cancer cells in vitro and in vivo. Nevertheless, no reports have disclosed whether DMC can affect the growth of human cervical cancer cells in vivo. Therefore we investigated the antitumor effects of DMC on a HeLa cell xenograft model in nude mice in this study. Materials and methods Twenty-four nude mice were subcutaneously injected with HeLa cells. All mice were randomly divided into control, low-dose DMC (30 mg/kg), and high-dose DMC (50 mg/kg) groups and individual mice were treated intraperitoneally accordingly every 2 days. Results DMC significantly reduced tumor weights and volumes of HeLa cell xenografts in mice, indicating the suppression of growth of xenograft tumors. Conclusion These effects and findings might provide evidence for investigating the potential use of DMC as an anti-cervical cancer drug in the future.

Cantharidin (CTD), a sesquiterpenoid bioactive substance, has been reported to exhibit anticancer activity against various types of cancer cells. The aim of the present study was to investigate the apoptosis effects and the underlying mechanisms of CTD on osteosarcoma U-2 OS cells. Results showed that CTD induced cell morphologic changes, reduced total viable cells, induced DNA damage, and G2/M phase arrest. CTD increased the production of reactive oxygen species and Ca2+, and elevated the activities of caspase-3 and −9, but decreased the level of mitochondrial membrane potential. Furthermore, CTD increased the ROS- and ER stress-associated protein expressions and increased the levels of pro-apoptosis-associated proteins, but decreased that of anti-apoptosis-associated proteins. Based on these observations, we suggested that CTD decreased cell number through G2/M phase arrest and the induction of cell apoptosis in U-2 OS cells and CTD could be a potential candidate for osteosarcoma treatments.

Lupeol, one of the common components from the fruits and natural foods, has been reported to exert antitumor activities in many human cancer cell lines; however, its effects on osteosarcoma cell metastasis were not elucidated. In the present study, lupeol at 10–25 μM induced cell morphological changes and decreased total viable cell number in U-2 OS cells. Lupeol (5–15 μM) suppressed cell mobility, migration, and invasion by wound healing and transwell chamber assays, respectively. Lupeol inhibited the activities of MMP-2 and −9 in U-2 OS cells by gelatin zymography assay. Lupeol significantly decreased PI3K, pAKT, β-catenin, and increased GSK3β. Furthermore, lupeol decreased the expressions of Ras, p-Raf-1, p-p38, and β-catenin. Lupeol also decreased uPA, MMP-2, MMP-9, and N-cadherin but increased VE-cadherin in U-2 OS cells. Based on these observations, we suggest that lupeol can be used in anti-metastasis of human osteosarcoma cells in the future.

Gastric cancer has a poor prognosis; once cancer has metastasized, it can easily lead to patient death. Melittin is one of the major components extracted from the bee venom. It has been shown that melittin emerges antitumor activities against many human cancer cell lines. Our results indicated that melittin at 0.2-0.5 µm significantly reduced total cell viability in human gastric cancer AGS cells. At low concentrations (0.05-0.15 µm), melittin displayed antimetastasis effects and inhibited cell adhesion and colony formation. Besides, it inhibited cell motility and suppressed cell migration and invasion. Melittin inhibited the activities of MMP-2 and MMP-9 and the integrity of cell membrane in AGS cells. Furthermore, Western blotting results showed that melittin decreased the protein expressions of Wnt/BMP and MMP-2 signaling pathways. Based on these observations, melittin inhibited cell migration and invasion of AGS cells through multiple signaling pathways. It may be used to treat metastasized gastric cancers in the future.

Background: Tetrandrine, a bis-benzylisoquinoline alkaloid, induces apoptosis of many types of human cancer cell. Hydrogen peroxide (H(2)O(2)) is a reactive oxygen species inducer; however, there are no reports to show whether pre-treatment of tetrandrine with H(2)O(2) induces more cell apoptosis than H(2)O(2) alone. Thus, the present study investigated the effects of tetrandrine on H(2)O(2)-induced cell apoptosis of human keratinocytes, HaCaT, in vitro. Materials and Methods: HaCaT cells were pre-treated with and without tetrandrine for 1 h, and then treated with H(2)O(2) for examining cell morphological changes and cell viability using contrast-phase microscopy and propidium iodide (PI) exclusion assay, respectively. Cells were measured apoptotic cell death by using annexin V/PI double staining and further analyzed by flow cytometer. Cells were further assessed for DNA condensation using 2-(4-amidinophenyl)-6-indolecarbamidine staining. Western blotting was used to measure expression of apoptosis-associated proteins and confocal laser microscopy was used to measure the protein expression and nuclear translocation from the cytoplasm to nuclei. Results: Pre-treatment of tetrandrine for 1 h and treatment with H(2)O(2) enhanced H(2)O(2)-induced cell morphological changes and reduced cell viability, whilst increasing apoptotic cell death and DNA condensation. Furthermore, tetrandrine significantly increased expression of reactive oxygen species-associated proteins such as superoxide dismutase (Cu/Zn) and superoxide dismutase (Mn) but significantly reduced the level of catalase, which was also confirmed by confocal laser microscopy. It also increased expression of DNA repair-associated proteins ataxia telangiectasia mutated, ataxia-telangectasia and Rad3-related, phospho-P53, P53 and phosphorylated histone H2AX, and of pro-apoptotic proteins BCL2 apoptosis regulator-associated X-protein, caspase-3, caspase-8, caspase-9 and poly ADP ribose polymerase in HaCaT cells. Conclusion: These are the first and novel findings showing tetrandrine enhances H(2)O(2)-induced apoptotic cell death of HaCaT cells and may provide a potent approach for the treatment of proliferated malignant keratinocytes.

Demethoxycurcumin (DMC), one of the derivatives of curcumin, has been shown to induce apoptotic cell death in many human cancer cell lines. However, there is no available information on whether DMC inhibits metastatic activity in human glioblastoma cancer cells in vitro.DMC at 1.0-3.0 μM significantly decreased the proliferation of GBM 8401 cells; thus, we used 2.0 μM for further investigation regarding anti-metastatic activity in human glioblastoma GBM 8401 cells.The internalized amount of DMC has reached the highest level in GBM 8401 cells after 3 h treatment. Wound healing assay was used to determine cell mobility and results indicated that DMC suppressed cell movement of GBM 8401 cells. The transwell chamber assays were used for measuring cell migration and invasion and results indicated that DMC suppressed cell migration and invasion in GBM 8401 cells. Gelatin zymography assay was used to examine gelatinolytic activity (MMP-2) in conditioned media of GBM 8401 cells treated by DMC and results demonstrated that DMC significantly reduced MMP-2 activity. Western blotting showed that DMC reduced the levels of p-EGFRDMC inhibited cancer cell migration and invasion through inhibition of PI3K/Akt and NF-κB signaling pathways in GBM 8401 cells. We suggest that DMC may be used as a novel anti-metastasis agent for the treatment of human glioblastoma cancer in the future.

Background/aim Maslinic acid, a natural plant-derived triterpenoid compound, exhibits pharmacological activities, including anti-cancer. In the present study, we investigated the cytotoxic effects of maslinic acid on human cervical cancer HeLa cells in vitro and further investigated the molecular mechanism of maslinic acid-induced DNA damage and repair. Materials and methods Cell viability was measured by flow cytometry. DNA condensation (apoptotic cell death), DNA damage, and DNA fragmentation (DNA ladder) were assayed by DAPI staining, comet assay, and agarose gel electrophoresis, respectively. The expression of DNA damage and repair proteins was assayed by western blotting. Results Maslinic acid decreased total cell viability and induced DNA condensation, damage, and fragmentation in HeLa cells. Furthermore, maslinic acid elevated the levels of p-ATMSer1981, p-ATRSer428, p53, p-p53Ser151, p-H2A.XSer139, BRCA1 and PARP at 30-40 μM. However, it decreased the levels of DNA-PK and MGMT. Conclusion Maslinic acid reduced the number of viable HeLa cells by inducing DNA damage and altering the expression of proteins involved in DNA damage and repair.

The objective of this study was to investigate the effects of tetrandrine (TET) on cell migration and invasion of nasopharyngeal carcinoma NPC-TW 039 cells in vitro. TET at 1-10 μM did not change cell morphology and also did not decrease the total cell viability and proliferation in NPC-TW 039 cells. It decreased the cell mobility based on decreased wound closure in NPC-TW 039 cells by wound healing assay. TET suppressed the cell migration and invasion using transwell system. TET reduced MMP-2 activities at 1-10 μM and these effects are in dose-dependently. After exposed to various treatments, TET decreased the levels of p-ERK, p-JNK, p-p38, RhoA, and NF-κB at 48 hr. Based on these findings, we may suggest TET-inhibited cell migration and invasion of NPC-TW 039 cells via the suppression of MAPK and RhoA signaling pathways for inhibiting the MMP-2 and -9 expression in vitro. PRACTICAL APPLICATIONS: Tetrandrine (TET), a bis-benzylisoquinoline alkaloid, is obtained from the dried root of Stephania tetrandra. TET has been shown to induce cancer cell apoptosis on human cancer cells but its anti-metastasis effect on cell migration and invasion of nasopharyngeal carcinoma cells has not been investigated. Our results showed that TET significantly repressed the cell mobility, migration, and invasion of NPC-TW 039 cells in vitro that involved in inhibiting RhoA, Ras accompanying with p38/MAPK signaling pathway. We conclude that TET may be the anticancer agents for nasopharyngeal carcinoma therapy in the future.

The aim of this study was to investigate the effects of bufalin on human nasopharyngeal carcinoma NPC-TW 076 cells in vitro. Bufalin is a cardiotonic steroid and a key active ingredient of the Chinese medicine ChanSu. The extracts of Chansu are used for various cancer treatments in China. In the present study, bufalin induced cell morphological changes, decreased total cell viability and induced G2/M phase arrest of cell cycle in NPC-TW 076 cells. Results also indicated that bufalin induced chromatin condensation (cell apoptosis) and DNA damage by DAPI staining and comet assay, respectively. The induced apoptotic cell death was further confirmed by annexin-V/PI staining assay. In addition, bufalin also increased ROS and Ca[Formula: see text] production and decreased the levels of [Formula: see text]. Furthermore, the alterations of ROS, ER stress and apoptosis associated protein expressions were investigated by Western blotting. Results demonstrated that bufalin increased the expressions of ROS associated proteins, including SOD (Cu/Zn), SOD2 (Mn) and GST but decreased that of catalase. Bufalin increased ER stress associated proteins (GRP78, IRE-1[Formula: see text], IRE-1[Formula: see text], caspase-4, ATF-6[Formula: see text], Calpain 1, and GADD153). Bufalin increased the pro-apoptotic proteins Bax, and apoptotic associated proteins (cytochrome c, caspase-3, -8 and -9, AIF and Endo G) but reduced anti-apoptotic protein Bcl-2 in NPC-TW 076 cells. Furthermore, bufalin elevated the expressions of TRAIL-pathway associated proteins (TRAIL, DR4, DR5, and FADD). Based on these findings, we suggest bufalin induced apoptotic cell death via caspase-dependent, mitochondria-dependent and TRAIL pathways in human nasopharyngeal carcinoma NPC-TW 076 cells.

Background/aim Evidence has indicated that fisetin induces cytotoxic effects in human cancer cell lines, including the inhibition of cell migration and invasion, however, the exact molecular mechanism of action of fisetin in human osteosarcoma cells remains unclear. Materials and methods The anti-metastatic mechanisms of fisetin in human osteosarcoma U-2 OS cells were investigated in vitro. Results Fisetin reduced the viability of cells at different concentrations (2.5, 5 and 10 μM) as measured by flow cytometric assay. Fisetin suppressed cell mobility, migration and invasion of U-2 OS cells, as shown by wound healing assay and transwell filter chambers, respectively. The gelatin zymography assay showed that fisetin inhibited MMP-2 activity in U-2 OS cells. Results from western blotting indicated that fisetin reduced the levels of pEGFR, SOS-1, GRB2, Ras, PKC, p-ERK1/2, p-JNK, p-p-38, VEGF, FAK, RhoA, PI3K, p-AKT, NF-ĸB, uPA, MMP-7, MMP-9, and MMP-13, but increased GSK3β and E-cadherin in U-2 OS cells after 48 h of treatment. Conclusion Fisetin can be used in the future, as a target for the treatment of metastasis of human osteosarcoma cells.

Genistein (GEN) has been shown to induce apoptotic cell death in various human cancer cells. L-asparaginase (Asp), a clinical drug for leukemia, has been shown to induce cell apoptosis in leukemia cells. No available information concerning GEN combined with Asp increased the cell apoptosis compared to GEN or Asp treatment alone. The objective of this study is to evaluate the anti-leukemia activity of GEN combined with Asp on human leukemia HL-60 cells in vitro. The cell viability, the distribution of cell cycle, apoptotic cell death, and the level of ΔΨm were examined by flow cytometric assay. The expressions of apoptosis-associated proteins were measured by western blotting. GEN combined with Asp revealed a more significant decrease in total viable cells and induced a higher percentage of G2/M phase arrest, DNA damage, and cell apoptosis than that of GEN or Asp treatment only in HL-60 cells. Furthermore, the combined treatments (GEN and Asp) showed a higher decrease in the level of ΔΨm than that of GEN or Asp treatment only. These results indicated that GEN combined with Asp induced mitochondria dysfunction by disrupting the mitochondrial membrane potential. The results from western blotting demonstrated that the treatment of GEN combined with Asp showed a higher increase in the levels of Bax and Bak (pro-apoptotic proteins) and an active form of caspase-3 and a higher decrease in Bcl-2 (anti-apoptotic protein) than that of GEN or Asp treatment alone. GEN significantly enhances the efficiency of Asp on cytotoxic effects (the induction of apoptosis) in HL-60 cells.

The aim of the present study was to investigate the cytotoxic effects of bufalin on SCC-4 human tongue cancer cells. Cell morphological changes and viability were examined using phase contrast microscopy and flow cytometry, respectively. The results indicated that bufalin induced morphological changes and reduced total viable cells. Apoptotic cell death was analyzed by DAPI staining and DNA gel electrophoresis; the results revealed that bufalin induced cell apoptosis. Levels of reactive oxygen species (ROS), Ca2+, nitric oxide (NO) and mitochondrial membrane potential (ΔΨm) were measured by flow cytometry, and bufalin was observed to increase Ca2+ and NO production, decrease the ΔΨm and reduce ROS production in SCC-4 cells. In addition, western blotting was performed to detect apoptosis-associated protein expression. The results demonstrated that bufalin reduced the expression of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) and increased the expression of the pro-apoptotic protein, Bcl-2-associated X protein. However, bufalin treatment also increased the expression of other apoptosis-associated proteins such as apoptosis-inducing factor and endonuclease G in SCC-4 cells. Based on these findings, bufalin may induce apoptotic cell death via mitochondria-dependent pathways in human tongue cancer SCC-4 cells.

Gypenosides (Gyp), the primary components of Gynostemma pentaphyllum Makino, have long been used as a Chinese herbal medicine. In the present study, the effects of Gyp on cell viability, the cell cycle, cell apoptosis, DNA damage and chromatin condensation were investigated in vitro using human oral cancer HSC-3 cells. The results of the present study indicated that Gyp induces cell death, G2/M phase arrest and apoptosis in HSC-3 cells in a dose-dependent manner. It was also demonstrated that Gyp decreased the depolarization of mitochondrial membrane potential in a time-dependent manner. A cDNA microarray assay was performed and the results indicated that a number of genes were upregulated following Gyp treatment. The greatest increase was a 75.42-fold increase in the expression of GTP binding protein in skeletal muscle. Levels of the following proteins were also increased by Gyp: Serpine peptidase inhibitor, clade E, member 1 by 20.25-fold; ras homolog family member B by 18.04-fold, kelch repeat and BTB domain containing 8 by 15.22-fold; interleukin 11 by 14.96-fold; activating transcription factor 3 by 14.49-fold; cytochrome P450, family 1 by 14.44-fold; ADP-ribosylation factor-like 14 by 13.88-fold; transfer RNA selenocysteine 2 by 13.23-fold; and syntaxin 11 by 13.08-fold. However, the following genes were downregulated by GYP: Six-transmembrane epithelial antigen of prostate family member 4, 14.19-fold; γ-aminobutyric acid A receptor by 14.58-fold; transcriptional-regulating factor 1 by 14.69-fold; serpin peptidase inhibitor, clade B, member 13 by 14.71-fold; apolipoprotein L 1 by 14.85-fold; follistatin by 15.22-fold; uncharacterized LOC100506718; fibronectin leucine rich transmembrane protein 2 by 15.61-fold; microRNA 205 by 16.38-fold; neuregulin 1 by 19.69-fold; and G protein-coupled receptor 110 by 22.05-fold. These changes in gene expression illustrate the effects of Gyp at the genetic level and identify potential targets for oral cancer therapy.

Oral cancer is one of the cancer-related diseases in human populations and its incidence rates are rising worldwide. Fisetin, a flavonoid from natural products, has been shown to exhibit anticancer activities in many human cancer cell lines but the molecular mechanism of fisetin-induced apoptosis in human oral cancer cells is still unclear; thus, in this study, we investigated fisetin-induced cell death and associated signal pathways on human oral cancer SCC-4 cells in vitro. We examined cell morphological changes, total viable cells, and cell cycle distribution by phase contrast microscopy and flow cytometry assays. Reactive oxygen species (ROS), Ca2+ , mitochondria membrane potential (ΔΨm ), and caspase-8, -9, and -3 activities were also measured by flow cytometer. Results indicate that fisetin induced cell death through the cell morphological changes, caused G2/M phase arrest, induction of apoptosis, promoted ROS and Ca2+ production, and decreased the level of ΔΨm and increased caspase-3, -8, and -9 activities in SCC-4 cells. DAPI staining and DNA gel electrophoresis were also used to confirm fisetin-induced cell apoptosis in SCC-4 cells. Western blotting also found out that Fisetin increased the proapoptotic proteins such as Bax and Bid and decreased the antiapoptotic proteins such as Bcl-2. Furthermore, results also showed that Fisetin increased the cytochrome c, AIF, and Endo G release from mitochondria in SCC-4 cells. We also used ATF-6α, ATF-6β, GADD153, and GRP78 which indicated that fisetin induced cell death through ER stress. Based on those observations, we suggest that fisetin induced cell apoptosis through ER stress, mitochondria-, and caspase-dependent pathways.

Cantharidin (CTD), a sesquiterpenoid bioactive substance, has been reported to exhibit anticancer activity against various types of cancer cells. The aim of the present study was to investigate the apoptosis effects and the underlying mechanisms of CTD on osteosarcoma U-2 OS cells. Results showed that CTD induced cell morphologic changes, reduced total viable cells, induced DNA damage, and G

Lupeol, one of the common components from the fruits and natural foods, has been reported to exert antitumor activities in many human cancer cell lines; however, its effects on osteosarcoma cell metastasis were not elucidated. In the present study, lupeol at 10-25 μM induced cell morphological changes and decreased total viable cell number in U-2 OS cells. Lupeol (5-15 μM) suppressed cell mobility, migration, and invasion by wound healing and transwell chamber assays, respectively. Lupeol inhibited the activities of MMP-2 and -9 in U-2 OS cells by gelatin zymography assay. Lupeol significantly decreased PI3K, pAKT, β-catenin, and increased GSK3β. Furthermore, lupeol decreased the expressions of Ras, p-Raf-1, p-p38, and β-catenin. Lupeol also decreased uPA, MMP-2, MMP-9, and N-cadherin but increased VE-cadherin in U-2 OS cells. Based on these observations, we suggest that lupeol can be used in anti-metastasis of human osteosarcoma cells in the future.

Cervical cancer is considered poorly chemo-sensitive in women and its treatment remains unsatisfactory. Cyperus rotundus is used in Chinese medicine as a therapeutic agent for women's disease. The effects and molecular mechanisms of the ethanol extraction of C. rotundus (CRE) on cervical cancer remain unclear. We aimed to explore the mechanisms and genetic influence of CRE on cervical cancer.HeLa, human cervical cancer cells were treated with various doses of CRE and changes in cell morphology and cell viability were assessed using microscopy and flow cytometry. Finally, we performed a microarray analysis to scan related genes.The treatment of CRE on HeLa cells caused morphological changes and induced chromatin condensation. DNA microarray analysis showed that CRE led to up-regulation of 449 genes and down-regulation of 484 genes, which were classified in several interaction pathways.CRE changed HeLa cell morphology and induced gene expression which associated with apoptosis and cell-cycle arrest. These results provide important information at the transcription level for targeting treatments of human cervical cancer.

Casticin, a polymethoxyflavone, has been demonstrated to possess anticancer activities, yet no study has shown in detail that casticin induces DNA damage in lung cancer cells. The purpose of this study was to investigate the possible molecular mechanisms of casticin which induce DNA damage and nuclear condensation in murine melanoma cancer B16F10 cells. In this study, by examining and capturing images using phase contrast microscopy, we found that casticin induced cell morphological changes. Moreover, it decreased the total number of viable cells which was measured by flow cytometry. Casticin-induced DNA damage and nuclear DNA condensation were measured by DAPI staining, respectively. Western blotting indicated that casticin decreased the protein levels of O6‑methylguanine-DNA methyltransferase (MGMT), breast cancer 1, early onset (BRCA1), mediator of DNA damage checkpoint 1 (MDC1), DNA-dependent protein kinase (DNA-PK) but increased phospho-p53 tumor suppressor protein (p-p53), phospho-ataxia telangiectasia mutated kinase (p-ATM), phospho-histone H2A.X (Ser139) and poly(ADP-ribose) polymerase (PARP) in the B16F10 cells. Furthermore, we used confocal laser system microscopy to examine the protein expression levels and we found that casticin increased the expression of p-p53 and p-H2A.X in the B16F10 cells. Collectively, casticin induced DNA damage and affected DNA repair proteins in the B16F10 cells in vitro.

Background/Aim: Evidence has indicated that fisetin induces cytotoxic effects in human cancer cell lines, including the inhibition of cell migration and invasion, however, the exact molecular mechanism of action of fisetin in human osteosarcoma cells remains unclear. Materials and Methods: The anti-metastatic mechanisms of fisetin in human osteosarcoma U-2 OS cells were investigated in vitro. Results: Fisetin reduced the viability of cells at different concentrations (2.5, 5 and 10 μM) as measured by flow cytometric assay. Fisetin suppressed cell mobility, migration and invasion of U-2 OS cells, as shown by wound healing assay and transwell filter chambers, respectively. The gelatin zymography assay showed that fisetin inhibited MMP-2 activity in U-2 OS cells. Results from western blotting indicated that fisetin reduced the levels of pEGFR, SOS-1, GRB2, Ras, PKC, p-ERK1/2, p-JNK, p-p-38, VEGF, FAK, RhoA, PI3K, p-AKT, NF-ĸB, uPA, MMP-7, MMP-9, and MMP-13, but increased GSK3β and E-cadherin in U-2 OS cells after 48 h of treatment. Conclusion: Fisetin can be used in the future, as a target for the treatment of metastasis of human osteosarcoma cells.

Numerous studies support the use of herbal medicine or natural products for chemotherapy in human cancers. Reports have associated curcumin (CUR), dimethoxy curcumin (DMC) and bisdemethoxycurcumin (BDMC) with numerous biological activities including anticancer activities, but no available information have shown that these induced apoptotic cell death and autophagy in human oral cancer cells. In the present study, we investigated the effect of CUR, DMC and BDMC on the cell viability, apoptotic cell death, reactive oxygen species (ROS), Ca[Formula: see text], mitochondria membrane potential (MMP) and caspase activities using flow cytometry assay and autophagy by monodansylcadaverine (MDC) and acridine orange (AO) staining in human oral cancer SAS cells. Results indicated that CUR, DMC and BDMC decreased total viable cell number through the induction of cell autophagy and apoptosis in SAS cells. Cells were pretreated with N-acetyl-cysteine (NAC), 3-methyladenine (3MA), rapamycin and carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoro-methylketone (Z-VAD-fmk) and then were treated with CUR, DMC and BDMC that led to increased total viable cell number when compared to CUR, DMC and BDMC treatments only. Results indicated induced apoptotic cell death through ROS, mitochondria-dependent pathway and induction of cell autophagy. Based on those observations, we suggest that CUR, DMC and BDMC could be used as a potential anticancer agent in human oral cancer.

Tetrandrine (TET) has been reported to induce anti-cancer activity in many human cancer cells and also to inhibit cancer cell migration and invasion. However, there are no reports to show TET inhibits cell migration and invasion in human brain glioblastoma multiforme GBM 8401 cells. In this study, we investigated the anti-metastasis effects of TET on GBM 8401 cells in vitro. Under sub-lethal concentrations (from 1, 5 up to 10 μM), TET significantly inhibited cell mobility, migration and invasion of GBM 8401 cells that were assayed by wound healing and Transwell assays. Gelatin zymography assay showed that TET inhibited MMP-2 activity in GBM 8401 cells. Western blotting results indicated that TET inhibited several key metastasis-related proteins, such as p-EGFR(Tyr1068) , SOS-1, GRB2, Ras, p-AKT(Ser473) and p-AKT(Thr308) , NF-κB-p65, Snail, E-cadherin, N-cadherin, NF-κB, MMP-2 and MMP-9 that were significant reduction at 24 and 48 hours treatment by TET. TET reduced MAPK signaling associated proteins such as p-JNK1/2 and p-c-Jun in GBM 8401 cells. The electrophoretic mobility shift (EMSA) assay was used to investigate NF-κB and DNA binding was reduced by TET in a dose-dependently. Based on these findings, we suggested that TET could be used in anti-metastasis of human brain glioblastoma multiforme GBM 8401 cells in the future.

BACKGROUND/AIM Demethoxycurcumin (DMC), one of the curcuminoids present in turmeric, has been shown to induce cell death in many human cancer cell lines, however, there has not been any investigation on whether DMC inhibits metastatic activity in human cervical cancer cells in vitro. In the present study, DMC at 2.5-15 μM decreased cell number, thus, we used IC20 (7.5 μM) for further investigation of its anti-metastatic activity in human cervical cancer HeLa cells. MATERIALS AND METHODS The wound healing, migration, invasion, zymography, and western blotting assays were used to investigate the effects of DMC on HeLa cells. RESULTS The wound healing assay was used to show that DMC suppressed cell movement of HeLa cells. Furthermore, the trans-well chamber assay was used to show that DMC suppressed HeLa cell migration and invasion. Gelatin zymography assay did not show any significant effects of DMC on the gelatinolytic activity (MMP-2 and -9) in conditioned media of HeLa cells treated by DMC. Western blotting showed that DMC significantly reduced protein levels of GRB2, MMP-2, ERK1/2, N-cadherin and Ras but increased the levels of E-cadherin and NF-κB in HeLa cells. Confocal laser microscopy indicated that DMC increased NF-κB in HeLa cells confirming the results from Western blotting. CONCLUSION DMC may be used as a novel anti-metastatic agent for the treatment of human cervical cancer in the future.

Background/aim Gefitinib is used to treat patients with lung cancer, but in some patients, the disease becomes gefitinib-resistant. Benzyl isothiocyanate (BITC), found in cruciferous vegetables, has shown anticancer activity in many human cancer cell lines. However, the effects of BITC on gefitinib-resistant NCI-H460 lung cancer cells in vitro have not been investigated. Materials and methods The effects of BITC on gefitinib-resistant NCI-H460 lung cancer cells were investigated in vitro. Flow cytometric assay was used for determining the total viable cell number, apoptotic cell death, the production of reactive oxygen species (ROS) and Ca2+, mitochondriaI membrane potential (Ψm) and caspase-3, -8 and -9 activities. Furthermore, 4', 6-diamidino-2-phenylindole staining was used to examine chromatin condensation in NCI-H460 and NCI-H460/G cells. Results BITC reduced total viable cell number via the induction of apoptotic cell death, that was also confirmed by annexin V/propidium iodide double staining assay. BITC increased ROS and Ca2+ production, reduced Ψm and increased caspase-3, -8 and -9 activities in both NCI-H460 and NCI-H460/G cells. Western blotting assay also showed that BITC increased expression of cleaved caspase-3 and -9, cytochrome c, BCL2-associated X protein, endonuclease G, poly (ADP-ribose) polymerase, growth arrest and DNA-damage protein 153, caspase-7 and activating transcription factor 6 alpha, but reduced apoptosis-inducing factor and caspase-9, BH3-interacting domain death agonist, calpain 1, glucose-regulated protein 78 and inositol requiring enzyme 1 alpha in NCI-H460/G cells. Conclusion BITC-induced apoptotic cell death appears to occur via caspase- and mitochondria-dependent pathways in both cell lines.

Demethoxycurcumin (DMC), one of the curcuminoids present in turmeric, has been shown to induce cell death in many human cancer cell lines, however, there has not been any investigation on whether DMC inhibits metastatic activity in human cervical cancer cells in vitro. In the present study, DMC at 2.5-15 μM decreased cell number, thus, we used ICThe wound healing, migration, invasion, zymography, and western blotting assays were used to investigate the effects of DMC on HeLa cells.The wound healing assay was used to show that DMC suppressed cell movement of HeLa cells. Furthermore, the trans-well chamber assay was used to show that DMC suppressed HeLa cell migration and invasion. Gelatin zymography assay did not show any significant effects of DMC on the gelatinolytic activity (MMP-2 and -9) in conditioned media of HeLa cells treated by DMC. Western blotting showed that DMC significantly reduced protein levels of GRB2, MMP-2, ERK1/2, N-cadherin and Ras but increased the levels of E-cadherin and NF-κB in HeLa cells. Confocal laser microscopy indicated that DMC increased NF-κB in HeLa cells confirming the results from Western blotting.DMC may be used as a novel anti-metastatic agent for the treatment of human cervical cancer in the future.

Cancer is the second cause of death in children. Osteosarcoma is the most common primary malignancy of solid bone cancer primarily affecting adolescents and young adults. In the Chinese population, the crude extract of Rheum palmatum L. (CERP) has been used for treating different diseases, including SARS, rheumatoid arthritis, coxsackievirus B3, and human colon cancer cell, pancreatic cancer. There are no reports on CERP and human osteosarcoma cells. The present study examined effects of CERP on cytotoxicity including cell cycle distribution and cell death (apoptosis) in U-2 OS human osteosarcoma cells. CERP significantly induced S phase arrest in U-2 OS cells in a dose-dependent. CERP produced DNA damage and DNA condensation. Other effects of CERP were stimulation of ROS and Ca(2+) , mitochondria impairment, and activation of caspase-3, -8, and -9. CERP increased the levels of Bax, Bak, Bad, cyclin B, Fas, PARP, GRP78, GADD153, AIF, Endo G, Calpain-2, p21, and p27, but decreased the levels of Bcl-2, BCL-X, XIAP, Akt, CDC25A, CDK2, Cyclin A, and Cyclin E of U-2 OS cells. It was also observed that CERP promoted the expression of AIF, Endo G, GADD153, and cytochrome c. These results indicate that CERP has anticancer effects in vitro and provide the foundation for in vivo studies of animal models of osteosarcoma. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 957-969, 2016.

Gefitinib has been used for cancer patients and curcumin (CUR), demethoxycurcumin (DMC), or bisdemethoxycurcumin (BDMC) also shown to induce cancer cell apoptosis. However, no report shows the combination of gefitinib with, CUR, DMC, or BDMC induce cell apoptosis and autophagy in human oral cancer cells. In this study, we investigated the effects of gefitinib with or without CUR, DMC, or BDMC co-treatment on the cell viability, apoptotic cell death, autophagy, mitochondria membrane potential (MMP), and caspase-3 activities by flow cytometry assay and autophagy by acridine orange (AO) staining in human oral cancer SAS cells. Results indicated that gefitinib co-treated with CUR, DMC, or BDMC decreased total viable cell number through the induction of cell apoptosis and autophagy and decreased the levels of MMP and increased caspase-3 activities in SAS cells. Western blotting indicated that gefitinib combined with CUR, DMC, or BDMC led to decrease Bcl-2 protein expression which is an antiapoptotic protein and to increase ATG5, Beclin 1, p62/SQSTM1, and LC3 expression that associated with cell autophagy in SAS cells. Gefitinib combined with CUR and DMC led to significantly reduce the tumor weights and volumes in SAS cell xenograft nude mice but did not affect the total body weights. Based on those observations, we suggest that the combination of gefitinib with CUR, DMC, and BDMC can be a potential anticancer agent for human oral cancer in future.

Polygonum cuspidatum is a natural plant that is used in traditional Chinese herbal medicine. The crude extract of Polygonum cuspidatum (CEPC) has numerous biological effects; however, there is a lack of studies on the effects of CEPC on immune responses in normal mice. The aim of the present study was to determine the in vivo effects of CEPC on immune responses in normal mice. CEPC (0, 50, 100, 150 and 200 mg/kg) was orally administered to BALB/c mice for three weeks, following which blood, liver, and spleen samples were collected. CEPC did not significantly affect the total body weight, or tissue weights of the liver or spleen, as compared with the control mice. CEPC increased the percentages of CD3 (T-cell marker), 11b (monocytes) and Mac-3 (macrophages) positive-cells, and reduced the percentage of CD19-positive cells (B-cell marker), as compared with the control mice. CEPC (100 mg/kg) stimulated macrophage phagocytosis of blood samples but did not affect macrophage phagocytosis in the peritoneum. Activity of the splenic natural killer cells was increased in response to CEPC (50 mg/kg) treatment. Furthermore, CEPC inhibited T- and B-cell proliferation when the cells were stimulated with concanavalin A and lipopolysaccharide, respectively.

Crude extract of Rheum palmatum L. (CERP) has been used to treat different diseases in the Chinese population for decades. In this study, we investigated the anti-metastasis effects of CERP on LS1034 human colorectal cancer cells in vitro and examined potential mechanisms of its effects. CERP significantly inhibited cell migration and invasion of LS1034 cells. We also found that CERP inhibited protein levels of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9), and cytosolic NF-kB p65, RHO A, ROCK 1. Furthermore, we found CERP inhibited protein levels of GRB2, SOS1, MKK7, FAK, Rho A, ROCK 1, VEGF, PKC, AKT, phosphor-AKT (Thr308), Cyclin D, iNOS, COX2, NF-kB p65, p-ERK1/2, p-JNK1/2, p-p38, p-c-jun, MMP-2, MMP-9, MMP-1, MMP-7, MMP-10, UPA and increased the protein level of Ras in LS1034 cells. In conclusion, our results suggest that CERP may be used as a novel anti-metastasis agent for the treatment of human colon cancer cells.

Curcumin (Cur), a natural product, has been shown to have anti-tumor activities in many human cancer cells. Gefitinib (Gef) is a clinical drug for cancer patients. However, there is no available information to show whether Gef/Cur nanoparticles (NPs) increased cell apoptosis and anti-tumor effects on xenograft mice model in vivo. In this study, γ-polyglutamic acid-coated nanoparticles loaded with Gef and Cur (γ-PGA-Gef/Cur NPs) were developed and its physicochemical properties and antitumor effects were investigated in vitro and in vivo. The γ-PGA-Gef/Cur NPs showed 548.5 ± 93.7 nm in diameter and −40.3 ± 3.87 mV on surface charge. The loading efficiencies of Gef and Cur were 89.5 and 100%, respectively. γ-PGA-Gef/Cur NPs could be internalized into SAS cells and significantly decreased total cell viability of SAS cells. Western blotting results indicated that both free Gef/Cur and γ-PGA-Gef/Cur NPs induced apoptotic cell death via caspase- and mitochondria-dependent pathways. In vivo studies indicated that treatments of PLGA NPs, free Gef/Cur, and γ-PGA-Gef/Cur NPs did not significantly affect appearances and bodyweights of mice. But the γ-PGA-Gef/Cur NPs significantly suppressed tumor size when comparing to free Gef/Cur-treated group. The nanoparticles developed in this study may be used as a potential therapy for oral cancer.

Irinotecan HCl (CPT-11) is an anticancer prodrug, but there is no available information addressing CPT-11-inhibited leukemia cells in in vitro and in vivo studies. Therefore, we investigated the cytotoxic effects of CPT-11 in promyelocytic leukemia HL-60 cells and in vivo and tumor growth in a leukemia xenograft model. Effects of CPT-11 on HL-60 cells were determined using flow cytometry, immunofluorescence staining, comet assay, real-time PCR, and Western blotting. CPT-11 demonstrated a dose- and time-dependent inhibition of cell growth, induction of apoptosis, and cell-cycle arrest at G0/G1 phase in HL-60 cells. CPT-11 promoted the release of AIF from mitochondria and its translocation to the nucleus. Bid, Bax, Apaf-1, caspase-9, AIF, Endo G, caspase-12, ATF-6b, Grp78, CDK2, Chk2, and cyclin D were all significantly upregulated and Bcl-2 was down-regulated by CPT-11 in HL-60 cells. Induction of cell-cycle arrest by CPT-11 was associated with changes in expression of key cell-cycle regulators such as CDK2, Chk2, and cyclin D in HL-60 cells. To test whether CPT-11 could augment antitumor activity in vivo, athymic BALB/c(nu/nu) nude mice were inoculated with HL-60 cells, followed by treatment with either CPT-11. The treatments significantly inhibited tumor growth and reduced tumor weight and volume in the HL-60 xenograft mice. The present study demonstrates the schedule-dependent antileukemia effect of CPT-11 using both in vitro and in vivo models. CPT-11 could potentially be a promising agent for the treatment of promyelocytic leukemia and requires further investigation.

Osteosarcoma is the most common primary bone malignancy and primarily occurs in adolescents and young adults. Crude extract of Rheum palmatum L. (CERP) has been used as a traditional Chinese medicine for different diseases and there is experimental evidence that it may have anti-cancer effects. However, there is no information showing that CERP can affect the mobility of human osteosarcoma cells. In this study we determined the effects of CERP on U-2 OS human osteosarcoma cells. We found that CERP significantly inhibited the migration and invasion of U-2 OS cells. CERP also reduced the activity of matrix metalloproteinase (MMP)-2 and MMP-9 and decreased expression levels of the proteins FAK, GRP78, PKC, HIF-1, SOS1, VEGF, PI3K, GRB2, Ras, p-ERK1/2, ERK1/2, p-p38, JNK1/2, p-JNK1/2, MEKK3, MKK7, PERK, p-PERK, iNOS, COX-2, NF-κB p65, IRE-1, UPA, and RhoA in U-2 OS cells. Confocal laser microscopy revealed that CERP decreased the expression of NF-κB p65, RhoA and Rock 1. These in vitro studies suggest that CERP may have novel anti-cancer actions in the treatment of osteosarcoma. Further studies including animal models of bone cancer are warranted.

Osteosarcoma is the most common primary malignancy of the bone cancers. In the Chinese population, the crude extract of Corni Fructus (CECF) has been used as Traditional Chinese medicine to treat several different diseases for hundreds of years. In the present study, effects of CECF on inhibition of migration and invasion in U-2 OS human osteosarcoma cells were examined. CECF significantly inhibited migration and invasion of U-2 OS human osteosarcoma cells. We also found that CECF inhibited activities of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9). CECF decreased protein levels of FAK, PKC, SOS1, MKK7, MEKK3, GRB2, NF-κB p65, COX-2, HIF-1α, PI3K, Rho A, ROCK-1, IRE-1α, p-JNK1/2, p-ERK1/2, p-p38, Ras, p-PERK, MMP-2, MMP-9, and VEGF in U-2 OS cells. Results of this study indicate that CECF may have potential as a novel anticancer agent for the treatment of osteosarcoma by inhibiting migration and invasion of cancer cells.