Biological effects and mechanisms of matrine and other constituents of Sophora flavescens in colorectal cancer.

The plant Sophora flavescens Ait. has been used in the clinical management of colorectal cancer (CRC). Its constituent compounds, notably the alkaloids matrine, oxymatrine, and sophoridine, have received considerable research attention in experimental models of CRC in vivo and in vitro. This review found that extracts of S. flavescens and/or its constituent compounds have been reported to inhibit CRC cell proliferation by inducing cell-cycle arrest at the G1 phase, inducing apoptosis via the intrinsic pathway, interfering in cancer metabolism, inhibiting metastasis and angiogenesis, regulating senescence and telomeres, regulating the tumour microenvironment and down-regulating cancer-related inflammation. In addition, matrine and oxymatrine reversed multi-drug resistance and enhanced the effects of chemotherapies. These anti-cancer effects were associated with regulation of several cellular signalling pathways including: MAPK/ERK, PI3K/AKT/mTOR, p38MAPK, NF-κB, Hippo/LATS2, TGF-ß/Smad, JAK/STAT3, RhoA/ROC, and Wnt/ ß-catenin pathways. These multiple actions in CRC suggest the alkaloids of S. flavescens may be therapeutic candidates for CRC management. Nevertheless, there remains considerable scope for future research into its flavonoid constituents, the effects of combinations of compounds, and the interaction between these compounds and anti-cancer drugs. In addition, more research is needed to investigate likely drug ligand-receptor interactions for each of the bioactive compounds.

Matrine combined with cisplatin synergistically inhibited urothelial bladder cancer cells via down-regulating VEGF/PI3K/Akt signaling pathway.

BACKGROUND: Cisplatin is one of the first-line drugs for urothelial bladder cancer (UBC) treatment. However, its considerable side effects and the emergence of drug resistance are becoming major limitations for its application. This study aimed to investigate whether matrine and cisplatin could present a synergistic anti-tumor effect on UBC cells. METHODS: Cell viability assay was used to assess the suppressive effect of matrine and cisplatin on the proliferation of the UBC cells. Wound healing assay and transwell assay were applied respectively to determine the migration and invasion ability of the cells. The distribution of cell cycles, the generation of reactive oxygen species (ROS) and the apoptosis rate were detected by flow cytometry (FCM). The expressions of the relative proteins in apoptotic signal pathways and the epithelial-mesenchymal transition (EMT) related genes were surveyed by western blotting. The binding modes of the drugs within the proteins were detected by CDOCKER module in DS 2.5. RESULTS: Both matrine and cisplatin could inhibit the growth of the UBC cells in a time- and dose-dependent manner. When matrine combined with cisplatin at the ratio of 2000:1, they presented a synergistic inhibitory effect on the UBC cells. The combinative treatment could impair cell migration and invasion ability, arrest cell cycle in the G1 and S phases, increase the level of ROS, and induce apoptosis in EJ and T24 cells in a synergistic way. In all the treated groups, the expressions of E-cadherin, ß-catenin, Bax, and Cleaved Caspase-3 were up-regulated, while the expressions of Fibronectin, Vimentin, Bcl-2, Caspase-3, p-Akt, p-PI3K, VEGFR2, and VEGF proteins were down-regulated, and among them, the combination of matrine and cisplatin showed the most significant difference. Molecular docking algorithms predicted that matrine and cisplatin could be docked into the same active sites and interact with different residues within the tested proteins. CONCLUSIONS: Our results suggested that the combination of matrine and cisplatin could synergistically inhibit the UBC cells' proliferation through down-regulating VEGF/PI3K/Akt signaling pathway, indicating that matrine may serve as a new option in the combinative therapy in the treatment of UBC.

Tanshinone IIA combined with adriamycin inhibited malignant biological behaviors of NSCLC A549 cell line in a synergistic way.

BACKGROUND: The study was designed to develop a platform to verify whether the extract of herbs combined with chemotherapy drugs play a synergistic role in anti-tumor effects, and to provide experimental evidence and theoretical reference for finding new effective sensitizers. METHODS: Inhibition of tanshinone IIA and adriamycin on the proliferation of A549, PC9 and HLF cells were assessed by CCK8 assays. The combination index (CI) was calculated with the Chou-Talalay method, based on the median-effect principle. Migration and invasion ability of A549 cells were determined by wound healing assay and transwell assay. Flow cytometry was used to detect the cell apoptosis and the distribution of cell cycles. TUNEL staining was used to detect the apoptotic cells. Immunofluorescence staining was used to detect the expression of Cleaved Caspase-3. Western blotting was used to detect the proteins expression of relative apoptotic signal pathways. CDOCKER module in DS 2.5 was used to detect the binding modes of the drugs and the proteins. RESULTS: Both tanshinone IIA and adriamycin could inhibit the growth of A549, PC9, and HLF cells in a dose- and time-dependent manner, while the proliferative inhibition effect of tanshinone IIA on cells was much weaker than that of adriamycin. Different from the cancer cells, HLF cells displayed a stronger sensitivity to adriamycin, and a weaker sensitivity to tanshinone IIA. When tanshinone IIA combined with adriamycin at a ratio of 20:1, they exhibited a synergistic anti-proliferation effect on A549 and PC9 cells, but not in HLF cells. Tanshinone IIA combined with adriamycin could synergistically inhibit migration, induce apoptosis and arrest cell cycle at the S and G2 phases in A549 cells. Both groups of the single drug treatment and the drug combination up-regulated the expressions of Cleaved Caspase-3 and Bax, but down-regulated the expressions of VEGF, VEGFR2, p-PI3K, p-Akt, Bcl-2, and Caspase-3 protein. Compared with the single drug treatment groups, the drug combination groups were more statistically significant. The molecular docking algorithms indicated that tanshinone IIA could be docked into the active sites of all the tested proteins with H-bond and aromatic interactions, compared with that of adriamycin. CONCLUSIONS: Tanshinone IIA can be developed as a novel agent in the postoperative adjuvant therapy combined with other anti-tumor agents, and improve the sensibility of chemotherapeutics for non-small cell lung cancer with fewer side effects. In addition, this experiment can not only provide a reference for the development of more effective anti-tumor medicine ingredients, but also build a platform for evaluating the anti-tumor effects of Chinese herbal medicines in combination with chemotherapy drugs.

Ellagic acid, a phenolic compound, exerts anti-angiogenesis effects via VEGFR-2 signaling pathway in breast cancer.

Anti-angiogenesis targeting VEGFR-2 has been considered as an important strategy for cancer therapy. Ellagic acid is a naturally existing polyphenol widely found in fruits and vegetables. It was reported that ellagic acid interfered with some angiogenesis-dependent pathologies. Yet the mechanisms involved were not fully understood. Thus, we analyzed its anti-angiogenesis effects and mechanisms on human breast cancer utilizing in-vitro and in-vivo methodologies. The in-silico analysis was also carried out to further analyze the structure-based interaction between ellagic acid and VEGFR-2. We found that ellagic acid significantly inhibited a series of VEGF-induced angiogenesis processes including proliferation, migration, and tube formation of endothelial cells. Besides, it directly inhibited VEGFR-2 tyrosine kinase activity and its downstream signaling pathways including MAPK and PI3K/Akt in endothelial cells. Ellagic acid also obviously inhibited neo-vessel formation in chick chorioallantoic membrane and sprouts formation of chicken aorta. Breast cancer xenografts study also revealed that ellagic acid significantly inhibited MDA-MB-231 cancer growth and P-VEGFR2 expression. Molecular docking simulation indicated that ellagic acid could form hydrogen bonds and aromatic interactions within the ATP-binding region of the VEGFR-2 kinase unit. Taken together, ellagic acid could exert anti-angiogenesis effects via VEGFR-2 signaling pathway in breast cancer.

LDH-A silencing suppresses breast cancer tumorigenicity through induction of oxidative stress mediated mitochondrial pathway apoptosis.

LDH-A, as the critical enzyme accounting for the transformation from pyruvate into lactate, has been demonstrated to be highly expressed in various cancer cells and its silencing has also been approved relating to increased apoptosis in lymphoma cells. In this study, we intend to investigate the correlation between LDH-A and other clinicopathological factors of breast cancer and whether LDH-A silencing could suppress breast cancer growth, and if so the potential mechanisms. 46 breast cancer specimens were collected to study the relation between LDH-A expression and clinicopathological characteristics including menopause, tumor size, node involvement, differentiation, and pathological subtypes classified by ER, PR, and Her-2. shRNAs were designed and applied to silence LDH-A expression in breast cancer cell lines MCF-7 and MDA-MB-231. The effects of LDH-A reduction on cancer cells were studied by a series of in vitro and in vivo experiments, including cell growth assay, apoptosis evaluation, oxidative stress detection, transmission electron microscopy observation, and tumor formation assay on nude mice. LDH-A expression was found to correlate significantly with tumor size and to be independent for other clinicopathological factors. LDH-A reduction resulted in an inhibited cancer cell proliferation, elevated intracellular oxidative stress, and induction of mitochondrial pathway apoptosis. Meanwhile, the tumorigenic ability of LDH-A deficient cancer cells was significantly limited in both breast cancer xenografts. The Ki67 positive cancer cells were significantly reduced in LDH-A deficiency tumor samples, while the apoptosis ratio was enhanced. Our results suggested that LDH-A inhibition might offer a promising therapeutic strategy for breast cancer.

Clonal expansions of cytotoxic T cells exist in the blood of patients with Waldenstrom macroglobulinemia but exhibit anergic properties and are eliminated by nucleoside analogue therapy.

T cells contribute to host-tumor interactions in patients with monoclonal gammopathies. Expansions of CD8(+)CD57(+) T-cell receptor Vbeta-positive (TCRVbeta(+))-restricted cytotoxic T-cell (CTL) clones are found in 48% of patients with multiple myeloma and confer a favorable prognosis. We now report that CTL clones with varying TCRVbeta repertoire are present in 70% of patients with Waldenström macroglobulinemia (WM; n = 20). Previous nucleoside analog (NA) therapy, associated with increased incidence of transformation to aggressive lymphoma, significantly influenced the presence of TCRVbeta expansions (chi(2) = 11.6; P < .001), as 83% of patients without (n = 6) and only 7% with (n = 14) TCRVbeta expansions had received NA. Clonality of CD3(+)CD8(+)CD57(+)TCRVbeta(+)-restricted CTLs was confirmed by TCRVbeta CDR3 size analysis and direct sequencing. The differential expression of CD3(+)CD8(+)CD57(+)TCRVbeta(+) cells was profiled using DNA microarrays and validated at mRNA and protein level. By gene set enrichment analysis, CTL clones expressed not only genes from cytotoxic pathways (GZMB, PRF1, FGFBP2) but also genes that suppress apoptosis, inhibit proliferation, arrest cell-cycle G1/S transition, and activate T cells (RAS, CSK, and TOB pathways). Proliferation tracking after stimulation confirmed their anergic state. Our studies demonstrate the incidence, NA sensitivity, and nature of clonal CTLs in WM and highlight mechanisms that cause anergy in these cells.

Osthole, a herbal compound, alleviates nucleus pulposus-evoked nociceptive responses through the suppression of overexpression of acid-sensing ion channel 3 (ASIC3) in rat dorsal root ganglion.

BACKGROUND: Osthole (Ost), a natural coumarin derivative, has been shown to inhibit many pro-inflammatory mediators and block voltage-gated Na+ channels. During inflammation, acidosis is an important pain inducer which activates nociceptors by gating depolarizing cationic channels, such as acid-sensing ion channel 3 (ASIC3). The aim of this study was to examine the effects of Ost on nucleus pulposus-evoked nociceptive responses and ASIC3 over-expression in the rat dorsal root ganglion, and to investigate the possible mechanism. MATERIAL/METHODS: Radicular pain was generated with application of nucleus pulposus (NP) to nerve root. Mechanical allodynia was evaluated using von Frey filaments with logarithmically incremental rigidity to calculate the 50% probability thresholds for mechanical paw withdrawal. ASIC3 protein expression in dorsal root ganglions (DRGs) was assessed with Western blot and immunohistochemistry. Membrane potential (MP) shift of DRG neurons induced by ASIC3-sensitive acid (pH6.5) was determined by DiBAC(4) (3) fluorescence intensity (F.I.). RESULTS: The NP-evoked mechanical hyperalgesia model showed allodynia for 3 weeks, and ASIC3 expression was up-regulated in DRG neurons, reaching peak on Day 7. Epidural administration of Ost induced a remarkable and prolonged antinociceptive effect, accompanied by an inhibition of over-expressed ASIC3 protein and of abnormal shift of MP. Amiloride (Ami), an antagonist of ASIC3, strengthened the antinociceptive effect of Ost. CONCLUSIONS: Up-regulation of ASIC3 expression may be associated with NP-evoked mechanical hyperalgesia. A single epidural injection of Ost decreased ASIC3 expression in DGR neurons and the pain in the NP-evoked mechanical hyperalgesia model. Osthole may be of great benefit for preventing chronic pain status often seen in lumbar disc herniation (LDH).

Modification of rat model of sciatica induced by lumber disc herniation and the anti-inflammatory effect of osthole given by epidural catheterization.

One of the most treatable causes of lower back pain and associated sciatica is lumbar disc herniation (LDH), which is characterized by rupture of the hard outer wall (annulus fibrosis) in a lumbar intervertebral disc. In the current study, we aimed to: (1) develop and characterize a rat model of sciatica induced by LDH, while introducing a novel method of epidural catheterization; (2) use this model to evaluate the effect of osthole on pain due to LDH, and (3) gain insight into the mechanisms through which osthole affects sciatica induced by LDH. The results indicate that our newly developed rat model maintained mechanical allodynia for 28 days without reduction. Moreover, cyclooxygenase-2 (COX-2) and nitric oxide synthase (NOS) were overexpressed in the associated inflammatory response, which is consistent with clinical manifestations of the disease. We then used this model to study the effect and mechanisms through which osthole affected pain due to LDH. Our study suggests that osthole is capable of reversing hyperalgesia due to LDH, potentially through modulation of activity of COX-2 and NOS, two important proteins for the exacerbation of pain due to LDH. Finally, a molecular modeling simulation showed that osthole has unique binding capabilities to both NOS and COX-2. As the model-induced mechanical hyperalgesia response was consistent, and the position of the catheter tip and the extension/spreading of the drug in the epidural space were reliable, this study developed an improved model to study remedies for sciatic pain. Moreover, our studies demonstrate that osthole may be a feasible treatment for the reduction of pain due to hyperalgesia.

Corn baked by burning coal triggered overexpression of osteopontin in hepatocytes of rats following fluorosis.

OBJECTIVE: To observe the expression of osteopontin (OPN) in hepatocytes of rats fed with corn baked by burning coal from fluorosis areas and a deficiency of calcium/protein intake following fluorosis. METHODS: A total of 48 Wistar rats as objects were randomly assorted into four groups: dose-free fluorine group, which were mainly fed with fluorine-free corn (56% structurally), dose-free fluorine with biased dietary group, which were fed with lower contents of protein (119.41 g/kg) and calcium (0.68 g/kg), high-dose fluorine group (fluorine contents: 104.2 mg/kg), and high-dose fluorine with biased dietary group. After 180 days of cultivation, the contents of fluorine in the bones of rats were tested for the assessment of construction of fluorosis animal model. And the expression of OPN in hepatocytes of rats in different groups was detected with immunohistochemistry and reverse transcription polymerase chain reaction. RESULTS: The present study validated the result that OPN was overexpressed in hepatocytes following fluorosis after oral intake of burning coal-baked corn. OPN was expressed most significantly in high fluorine with biased dietary group, and the high-fluorine group ranked the second most; and dose-free fluorine with biased dietary group ranked the third. The dose-free fluorine group expressed the least OPN. CONCLUSION: Overexpression of OPN in hepatocytes following fluorosis after excess fluorine intake was involved in liver damage process, which was enhanced by deficiency of calcium and protein intake. The results also demonstrated that the development of fluorosis in Guizhou province was correlated with local baking staple corn as a way of excess intake of fluorine and deficiency of calcium/protein intake.

Regulation of human pregnane X receptor and its target gene cytochrome P450 3A4 by Chinese herbal compounds and a molecular docking study.

The pregnane X receptor (PXR) plays a critical role in the regulation of human cytochrome P450 3A4 (CYP3A4) gene. In this study, we investigated the effect of an array of compounds isolated from Chinese herbal medicines on the activity of PXR using a luciferase reporter gene assay in transiently transfected HepG2 and Huh7 cells and on the expression of PXR and CYP3A4 in LS174T cells. Furthermore, molecular docking was performed to investigate the binding modes of herbal compounds with PXR. Praeruptorin A and C, salvianolic acid B, sodium danshensu, protocatechuic aldehyde, cryptotanshinone, emodin, morin, and tanshinone IIA significantly transactivated the CYP3A4 reporter gene construct in either HepG2 or Huh7 cells. The PXR mRNA expression in LS174T cells was significantly induced by physcion, protocatechuic aldehyde, salvianolic acid B, and sodium danshensu. However, epifriedelanol, morin, praeruptorin D, mulberroside A, tanshinone I, and tanshinone IIA significantly down-regulated the expression of PXR mRNA in LS174T cells. All the herbal compounds tested can be readily docked into the ligand-binding cavity of PXR mainly through hydrogen bond and aromatic interactions with Ser247, Gln285, His407, and Arg401. These findings suggest that herbal medicines can significantly regulate PXR and CYP3A4 and this has important implication in herb-drug interactions.

Multidrug resistance-associated proteins and implications in drug development.

1. The multidrug resistance-associated proteins (MRPs) belong to the ATP-binding cassette superfamily (ABCC family) of transporters that are expressed differentially in the liver, kidney, intestine and blood-brain barrier. There are nine human MRPs that transport a structurally diverse array of endo- and xenobiotics as well as their conjugates. 2. Multidrug resistance-associated protein 1 can be distinguished from MRP2 and MRP3 by its higher affinity for leukotriene C(4). Unlike MRP1, MRP2 functions in the extrusion of endogenous organic anions, such as bilirubin glucuronide and certain anticancer agents. In addition to the transport of glutathione and glucuronate conjugates, MRP3 has the additional capability of mediating the transport of monoanionic bile acids. 3. Both MRP4 and MRP5 are able to mediate the transport of cyclic nucleotides and confer resistance to certain antiviral and anticancer nucleotide analogues. Hereditary deficiency of MRP6 results in pseudoxanthoma elasticum. In the body, MRP6 is involved in the transport of glutathione conjugates and the cyclic pentapeptide BQ123. 4. Various MRPs show considerable differences in tissue distribution, substrate specificity and proposed physiological function. These proteins play a role in drug disposition and excretion and thus are implicated in drug toxicity and drug interactions. Increased efflux of natural product anticancer drugs and other anticancer agents mediated by MRPs from cancer cells is associated with tumour resistance. 5. A better understanding of the function and regulating mechanisms of MRPs could help minimize and avoid drug toxicity and unfavourable drug-drug interactions, as well as help overcome drug resistance.

The Synergistic Antitumor Effect of Tanshinone IIA Plus Adriamycin on Human Hepatocellular Carcinoma Xenograft in BALB/C Nude Mice and Their Influences on Cytochrome P450 CYP3A4 In Vivo.

OBJECTIVE: Hepatocellular carcinoma is one of the most common diseases that seriously threaten human life and health. In this study, we evaluated the inhibitory effect of tanshinone IIA (Tan IIA) combined with adriamycin (ADM) on human hepatocellular carcinoma and developed a platform to assess the function if Chinese herbal ingredients combined with chemotherapy drugs have synergistic antitumor effects in vivo. METHODS: Established animal model of human hepatocarcinoma HepG2 cell in nude mice. Mice were divided into model control group, Tan IIA group, ADM group, and Tan IIA + ADM group. The changes from general condition, weight, tumor volume, and inhibition rate were observed. The data were gathered from serum AST level and histopathological changes. The content and activity of cytochrome P450 were determined by spectrophotometric analysis. CYP3A4 protein expression was analyzed by western blotting. The binding model crystal structure of Tan IIA and ADM with pregnane X receptor (PXR) was evaluated by Discovery Studio 2.1. RESULTS: A combination of Tan IIA with ADM could improve life quality by relieving ADM toxicity, decreasing tumor volume, declining serum AST level, and improving liner pathological section in tumor-bearing mice. The inhibitory rates of Tan IIA, ADM, and cotreatment were 32.77%, 60.96%, and 73.18%, respectively. The Tan IIA group significantly enhanced the content of cytochrome b5, P450, and erythromycin-N-demethylase activity. CYP3A4 protein expression was enhanced obviously by the Tan IIA + ADM group. Virtual molecular docking showed that both Tan IIA and ADM could be stably docked with the same binding site of PXR but different interactions. CONCLUSIONS: Tan IIA in combination with ADM could improve the life quality in tumor-bearing mice and enhance the antitumor effect. The Tan IIA group increased the concentration of cytochrome P450 enzymes and activity. Combined Tan IIA with ADM could upregulate the CYP3A4 protein expression and make relevant interaction with protein PXR by virtual docking.

Matrine induces apoptosis in multiple colorectal cancer cell lines in vitro and inhibits tumour growth with minimum side effects in vivo via Bcl-2 and caspase-3.

BACKGROUND: The World Health Organization (WHO) reported that colorectal cancer (CRC) was the third most common cancer in men and the second in women, worldwide. Our previous meta-analysis found Sophora flavescens increased tumour response rate in randomised controlled trials of CRC. We hypothesised that its principal constituent matrine had exerted anti-tumour effects. PURPOSE: To elucidate its mechanisms of action we investigated the dose-related anti-tumour effects of matrine on four human CRC cell-lines: LS174T, Caco-2, SW1116 and RKO. In a LS174T xenografted tumour model in nude mice we assessed the effects of matrine and oxaliplatin on tumour volume, weight and morphology. Computer simulated dockings for target proteins were also conducted. METHODS AND DESIGN: Cell viability, cell cycle and apoptosis were measured by Cell Counting Kit-8 and flow cytometry, and Annexin V-FITC/PI double staining assay respectively. Western blot was performed to examine the expression of Bax, Bcl-2 and caspase-3 in the cells. The xenograft model and immunohistochemistry were used to investigate the effect of matrine in vivo. Oxaliplatin was set as positive control. Molecular docking was performed to predict the binding modes of matrine and oxaliplatin with target proteins using CDOCKER algorithm. RESULTS: Matrine inhibited proliferation of cancer cells in a dose- and time-dependent manner. Matrine induced cell-cycle arrest at G1/G0 phase, induced apoptosis and reduced expression of Bcl-2 and caspase-3 while up-regulating Bax and cleaved caspase-3 in the four CRC cells. In vivo, matrine significantly inhibited tumour growth without side effects on physical health compared to the negative (vehicle) control group. Mice in the oxaliplatin group lost vigour, became frail and lost weight. Expression of Bcl-2 in tumour tissue was lower and Bax expression was higher in the matrine-treated groups compared to the negative control. In computer-simulated docking, matrine successfully docked into active sites of Bcl-2 and caspase-3. CONCLUSION: Matrine inhibited growth of colorectal cancer cells in vitro and in vivo. A molecular mechanism was apoptosis induction via effects on Bcl-2, Bax and caspase-3. Moreover, matrine showed minimum side effects and may provide a candidate for the development of new therapies for colorectal cancer.

The antitumor effect of tanshinone IIA on anti-proliferation and decreasing VEGF/VEGFR2 expression on the human non-small cell lung cancer A549 cell line.

The effects of tanshinone IIA on the proliferation of the human non-small cell lung cancer cell line A549 and its possible mechanism on the VEGF/VEGFR signal pathway were investigated. The exploration of the interaction between tanshinone IIA and its target proteins provides a feasible platform for studying the anticancer mechanism of active components of herbs. The CCK-8 assay was used to evaluate the proliferative activity of A549 cells treated with tanshinone IIA (2.5-80 µmol/L) for 24, 48 and 72 h, respectively. Flow cytometry was used for the detection of cell apoptosis and cell cycle perturbation. VEGF and VEGFR2 expression were studied by Western blotting. The binding mode of tanshinone IIA within the crystal structure of the VEGFR2 protein was evaluated with molecular docking analysis by use of the CDOCKER algorithm in Discovery Studio 2.1. The CCK-8 results showed that tanshinone IIA can significantly inhibit A549 cell proliferation in a dose- and time-dependent manner. Flow cytometry results showed that the apoptosis rate of tested group was higher than the vehicle control, and tanshinone IIA-treated cells accumulated at the S phase, which was higher than the vehicle control. Furthermore, the expression of VEGF and VEGFR2 was decreased in Western blot. Finally, molecular docking analysis revealed that tanshinone IIA could be stably docked into the kinase domain of VEGFR2 protein with its unique modes to form H-bonds with Cys917 and π-π stacking interactions with Val848. In conclusion, tanshinone IIA may suppress A549 proliferation, induce apoptosis and cell cycle arrest at the S phase. This drug may suppress angiogenesis by targeting the protein kinase domains of VEGF/VEGFR2.

[Clinical observation on colquhounia root tablet in treating lipid metabolism disturbance secondary to nephrotic syndrome].

OBJECTIVE: To study the effect of Colquhounia root tablet (CRT) in treating nephrotic syndrome with sequential lipid metabolism disorder (NS-LMD). METHODS: The 96 patients with NS-LMD were randomly divided into two groups, the 60 cases in the treated group treated with CRT and the 36 cases in the control group treated with hormone or cytotoxic medicine. The curative effect and the related indexes were determined before and after treatment. RESULTS: After six months treatment, the general effective rate in the treated group was 88. 33%, which was markedly higher than that in the control group (69.44%, P < 0.05). The levels of the treated group in ameliorating lipid metabolism disorder and renal dysfunction were also higher than those in the control group (P < 0.05). CONCLUSION: CRT could improve NS-LMD, improve renal function, eliminate urinary protein and increase plasma albumin. It is highly effective with low toxicity and safe.

Baicalein decreases side population proportion via inhibition of ABCG2 in multiple myeloma cell line RPMI 8226 in vitro.

OBJECTIVE: To investigate the effect of baicalein on side population in human multiple myeloma cell line RPMI 8226 and the underlying molecular mechanisms in vitro and in silico. METHODS: MTT assay was applied to detect the anti-proliferation effect of baicalein. The detection of side population cells is based on the Hoechst 33342 exclusion assay technique and flow cytometric analysis. Western blotting assay was used to explore the expression of ABCG2 protein. Homology modeling and molecular docking were performed with Discovery Studio 2.1. RESULTS: Baicalein decreased both cell viability with IC50=168.5 µM and the proportion of SP cells in a dose-dependent manner. Correspondingly, it significantly decreased the expression level of ABCG2 protein. Baicalein also shared similar binding sites and modes with fumitremorgin C to the protein. CONCLUSIONS: Baicalein possessed novel anticancer properties, such as anti-proliferation and drug efflux inhibition in side population cells, which suggested its potential feature of targeting cancer stem cells of multiple myeloma.

Wogonin induces apoptosis in RPMI 8226, a human myeloma cell line, by downregulating phospho-Akt and overexpressing Bax.

AIMS: Wogonin is one of the major constituents derived from Scutellaria Baicalensis, which has been reported to inhibit cell growth and/or induce apoptosis in various cancer cell lines. We aim to investigate the anticancer effects and associated mechanisms of wogonin on human multiple myeloma cell line in vitro. MAIN METHODS: Effects of wogonin on the proliferation, cell cycle progression, and apoptosis of human myeloma cells were examined in vitro. The proteins associated with the biological effects of wogonin were analyzed by immunoblotting and immunocytochemical staining. In addition, the binding mode of wogonin within crystal structure of Akt1 protein was also evaluated by molecular docking analysis using the CDOCKER algorithm in Discovery Studio. KEY FINDINGS: Myeloma cell growth was attenuated by wogonin (70.4-352.0 µM) in a concentration-dependent manner. Cell cycle progression analysis and TUNEL assay showed that apoptosis was enhanced in wogonin-treated cells. Increased apoptosis was accompanied by decreased level of total-PARP, the arisen of PARP cleavage, significantly increased level of Bax protein and decreased level of Bcl-2 protein. Akt activity was suppressed and phosphorylation of Ser 473 residue was decreased in the wogonin-treated cells. Molecular docking analysis revealed wogonin could be stably docked into the ligand binding domain of Akt1 protein, and presented unique features of binding to Akt1, which indicated detailed interaction between wogonin and Akt signaling pathway. SIGNIFICANCE: As wogonin was effective in vitro in promotion of apoptosis of myeloma cell by Akt-modulated, Bax and Bcl-2 related intrinsic apoptotic pathway, wogonin may be a potential therapeutic agent against multiple myeloma.

Scutellaria extract decreases the proportion of side population cells in a myeloma cell line by down-regulating the expression of ABCG2 protein.

BACKGROUND AND AIMS: Scutellaria is one of the most popular traditional Chinese herbal remedies against various human diseases, including cancer. In this study, we examined the active effects of Scutellaria extract and its main flavonoid constituents on the proportion of side population cells within human multiple myeloma cell line RPMI8226 in vitro and explored the potential molecular mechanisms involved. MATERIALS AND METHODS: The contents of flavonoids in ethanolic extract of Scutellaria baicalensis Georgi were determined using high performance liquid chromatography. The antiproliferative effect of the ethanolic extract on RPMI-8226 was determined by CCK assay. Apoptosis was measured by annexin combining with propidium iodide in a flow cytometer. Cell cycle analysis was performed by propidium iodide staining in combination with flow cytometry analysis. Hoechst 33342 exclusion assay was used for the identification of side population within RPMI8226 cells. The expression of ABCG2 protein was assessed by Western blotting assay. RESULTS: The content of major flavonoids constitutents of Scutellaria extract was baicalin (10.2%), wogonoside (2.50%), baicalein (2.29%), and wogonin (0.99%), respectively. The crude Scutellaria extract did not show significant anti-proliferative effect, apoptosis induction and cell cycle arrest in RPMI-8226 within the concentrations of 1-75µg/mL. However, the ethanolic extract, baicalein, wogonin and baicalin reduced the side population cells in RPMI-8226, and data showed that baicalein and wogonin had stronger inhibitory effects. Correspondingly, they also exhibited significant effects on decreasing the expression level of ABCG2 protein in RPMI-8226 in vitro. CONCLUSIONS: Our results for the first time demonstrated a novel mechanism of action for Scutellaria extract and its main active flavonoids, namely targeting SP cells by modulating the expression of ABCG2 protein. This study provides an insight for new therapeutic strategies targeting cancer stem cells of multiple myeloma.

Evidence-based toxicity evaluation and scheduling of Chinese herbal medicines.

ETHNOPHARMACOLOGICAL RELEVANCE: While there is an increasing number of toxicity report cases and toxicological studies on Chinese herbal medicines, the guidelines for toxicity evaluation and scheduling of Chinese herbal medicines are lacking. AIM: The aim of this study was to review the current literature on potentially toxic Chinese herbal medicines, and to develop a scheduling platform which will inform an evidence-based regulatory framework for these medicines in the community. MATERIALS AND METHODS: The Australian and Chinese regulations were used as a starting point to compile a list of potentially toxic herbs. Systematic literature searches of botanical and pharmaceutical Latin name, English and Chinese names and suspected toxic chemicals were conducted on Medline, PubMed and Chinese CNKI databases. RESULTS: Seventy-four Chinese herbal medicines were identified and five of them were selected for detailed study. Preclinical and clinical data were summarised at six levels. Based on the evaluation criteria, which included risk-benefit analysis, severity of toxic effects and clinical and preclinical data, four regulatory classes were proposed: Prohibited for medicinal usage, which are those with high toxicity and can lead to injury or death, e.g., aristolochia; Restricted for medicinal usage, e.g., aconite, asarum, and ephedra; Required warning label, e.g., coltsfoot; and Over-the-counter herbs for those herbs with a safe toxicity profile. CONCLUSION: Chinese herbal medicines should be scheduled based on a set of evaluation criteria, to ensure their safe use and to satisfy the need for access to the herbs. The current Chinese and Australian regulation of Chinese herbal medicines should be updated to restrict the access of some potentially toxic herbs to Chinese medicine practitioners who are qualified through registration.

Pharmacophore, QSAR, and binding mode studies of substrates of human cytochrome P450 2D6 (CYP2D6) using molecular docking and virtual mutations and an application to chinese herbal medicine screening.

The highly polymorphic human cytochrome P450 2D6 (CYP2D6) metabolizes about 25% of currently used drugs. In this study, we have explored the interaction of a large number of substrates (n = 120) with wild-type and mutated CYP2D6 by molecular docking using the CDOCKER module. Before we conducted the molecular docking and virtual mutations, the pharmacophore and QSAR models of CYP2D6 substrates were developed and validated. Finally, we explored the interaction of a traditional Chinese herbal formula, Fangjifuling decoction, with CYP2D6 by virtual screening. The optimized pharmacophore model derived from 20 substrates of CYP2D6 contained two hydrophobic features and one hydrogen bond acceptor feature, giving a relevance ratio of 76% when a validation set of substrates were tested. However, our QSAR models gave poor prediction of the binding affinity of substrates. Our docking study demonstrated that 117 out of 120 substrates could be docked into the active site of CYP2D6. Forty one out of 117 substrates (35.04%) formed hydrogen bonds with various active site residues of CYP2D6 and 53 (45.30%) substrates formed a strong π-π interaction with Phe120 (53/54), with only carvedilol showing π-π interaction with Phe483. The active site residues involving hydrogen bond formation with substrates included Leu213, Lys214, Glu216, Ser217, Gln244, Asp301, Ser304, Ala305, Phe483, and Phe484. Furthermore, the CDOCKER algorithm was further applied to study the impact of mutations of 28 active site residues (mostly non-conserved) of CYP2D6 on substrate binding modes using five probe substrates including bufuralol, debrisoquine, dextromethorphan, sparteine, and tramadol. All mutations of the residues examined altered the hydrogen bond formation and/or aromatic interactions, depending on the probe used in molecular docking. Apparent changes of the binding modes have been observed with the Glu216Asp and Asp301Glu mutants. Overall, 60 compounds out of 130 from Fangjifuling decoction matched our pharmacophore model for CYP2D6 substrates. Fifty four out of these 60 compounds could be docked into the active site of CYP2D6 and 24 of 54 compounds formed hydrogen bonds with Glu216, Asp301, Ser304, and Ala305 in CYP2D6. These results have provided further insights into the factors that determining the binding modes of substrates to CYP2D6. Screening of high-affinity ligands for CYP2D6 from herbal formula using computational models is a useful approach to identify potential herb-drug interactions.