Characterization of the complete chloroplast genome of the medicinal herb Eleutherococcus nodiflorus and its phylogenetic implications.

Eleutherococcus nodiflorus (Dunn) S. Y. Hu is a momentous medicinal plant belonging to the Araliaceae family. In the current investigation, we determined the complete chloroplast genome of E. nodiflorus and analyzed the phylogenetic relationship among Eleutherococcus plants. The chloroplast genome of E. nodiflorus exhibited a typical quadripartite structure with a full length of 156,770 bp, including 133 genes, containing 88 protein-coding genes, 8 rRNA genes, 37 tRNA genes, and 1 presumed pseudogene (ycf1). The overall GC content observed was 37.95%, with the highest GC content of 43.02% found in the IR region. Comparative genome analysis revealed five highly variable regions among Eleutherococcus species, providing potential markers for further investigations on species identification and population genetics. A total of 44 small simple repeats were identified throughout the chloroplast genome of E. nodiflorus. The phylogenetic analysis indicated a sister relationship between E. nodiflorus and E. eleutheristylus, suggesting a close genetic relationship between the two Eleutherococcus plants. These results enhance the understanding of the plant evolution within Eleutherococcus plants and provide basic genetic resources for the development of species identification and investigation of population genetic diversity of the Eleutherococcus genus and Araliaceae.

The complete chloroplast genomes of Tetrastigma hemsleyanum (Vitaceae) from different regions of China: molecular structure, comparative analysis and development of DNA barcodes for its geographical origin discrimination.

BACKGROUND: Tetrastigma hemsleyanum is a valuable traditional Chinese medicinal plant widely distributed in the subtropical areas of China. It belongs to the Cayratieae tribe, family Vitaceae, and exhibited significant anti-tumor and anti-inflammatory activities. However, obvious differences were observed on the quality of T. hemsleyanum root from different regions, requiring the discrimination strategy for the geographical origins. RESULT: This study characterized five complete chloroplast (cp) genomes of T. hemsleynum samples from different regions, and conducted a comparative analysis with other representing species from family Vitaceae to reveal the structural variations, informative markers and phylogenetic relationships. The sequenced cp genomes of T. hemsleyanum exhibited a conserved quadripartite structure with full length ranging from 160,124 bp of Jiangxi Province to 160,618 bp of Zhejiang Province. We identified 112 unique genes (80 protein-coding, 28 tRNA and 4 rRNA genes) in the cp genomes of T. hemsleyanum with highly similar gene order, content and structure. The IR contraction/expansion events occurred on the junctions of ycf1, rps19 and rpl2 genes with different degrees, causing the differences of genome sizes in T. hemsleyanum and Vitaceae plants. The number of SSR markers discovered in T. hemsleyanum was 56-57, exhibiting multiple differences among the five geographic groups. Phylogenetic analysis based on conserved cp genome proteins strongly grouped the five T. hemsleyanum species into one clade, showing a sister relationship with T. planicaule. Comparative analysis of the cp genomes from T. hemsleyanum and Vitaceae revealed five highly variable spacers, including 4 intergenic regions and one protein-coding gene (ycf1). Furthermore, five mutational hotspots were observed among T. hemsleyanum cp genomes from different regions, providing data for designing DNA barcodes trnL and trnN. The combination of molecular markers of trnL and trnN clustered the T. hemsleyanum samples from different regions into four groups, thus successfully separating specimens of Sichuan and Zhejiang from other areas. CONCLUSION: Our study obtained the chloroplast genomes of T. hemsleyanum from different regions, and provided a potential molecular tracing tool for determining the geographical origins of T. hemsleyanum, as well as important insights into the molecular identification approach and and phylogeny in Tetrastigma genus and Vitaceae family.

Complete chloroplast genome of the medicinal herb Veronicastrum axillare (Sieb. et Zucc.) Yamazaki and the phylogenetic relationship analysis within the tribe Veroniceae.

Veronicastrum axillare (Sieb. et Zucc.) Yamazaki is a traditional medical plant with versatile biological activities. Here, we reported the complete chloroplast genome sequence of V. axillare with a total length of 152,691 bp, containing two IR regions of 25,765 bp each, an LSC region of 83,559 bp, and an SSC region of 17,602 bp. The genome encodes 131 genes, including 85 protein-coding genes, 37 tRNAs, eight rRNAs, and one pseudogene (ycf1). The overall GC content is 38.3%, with the highest content of 43.31% in IR region. Comparative analysis revealed 4 potential hotspots among V. axillare and other Veroniceae plants, providing potential markers for population investigations in the tribe Veroniceae. A total of 56 simple sequence repeats were identified in V. axillare. Phylogenetic analysis indicated a sister relationship between V. axillare and V. sibiricum, suggesting a close genetic relationship between the two Veronicastrum species. Our results provide basic genetic resources for investigating the evolutionary status of V. axillare within the tribe Veroniceae.

Complete chloroplast genome of Stephania tetrandra (Menispermaceae) from Zhejiang Province: insights into molecular structures, comparative genome analysis, mutational hotspots and phylogenetic relationships.

BACKGROUND: The Stephania tetrandra S. Moore (S. tetrandra) is a medicinal plant belonging to the family Menispermaceae that has high medicinal value and is well worth doing further exploration. The wild resources of S. tetrandra were widely distributed in tropical and subtropical regions of China, generating potential genetic diversity and unique population structures. The geographical origin of S. tetrandra is an important factor influencing its quality and price in the market. In addition, the species relationship within Stephania genus still remains uncertain due to high morphological similarity and low support values of molecular analysis approach. The complete chloroplast (cp) genome data has become a promising strategy to determine geographical origin and understand species evolution for closely related plant species. Herein, we sequenced the complete cp genome of S. tetrandra from Zhejiang Province and conducted a comparative analysis within Stephania plants to reveal the structural variations, informative markers and phylogenetic relationship of Stephania species. RESULTS: The cp genome of S. tetrandra voucher ZJ was 157,725 bp, consisting of a large single copy region (89,468 bp), a small single copy region (19,685 bp) and a pair of inverted repeat regions (24,286 bp each). A total of 134 genes were identified in the cp genome of S. tetrandra, including 87 protein-coding genes, 8 rRNA genes, 37 tRNA genes and 2 pseudogene copies (ycf1 and rps19). The gene order and GC content were highly consistent in the Stephania species according to the comparative analysis results, with the highest RSCU value in arginine (1.79) and lowest RSCU value in serine of S. tetrandra, respectively. A total of 90 SSRs have been identified in the cp genome of S. tetrandra, where repeats that consisting of A or T bases were much higher than that of G or C bases. In addition, 92 potential RNA editing sites were identified in 25 protein-coding genes, with the most predicted RNA editing sites in ndhB gene. The variations on length and expansion extent to the junction of ycf1 gene were observed between S. tetrandra vouchers from different regions, indicating potential markers for further geographical origin discrimination. Moreover, the values of transition to transversion ratio (Ts/Tv) in the Stephania species were significantly higher than 1 using Pericampylus glaucus as reference. Comparative analysis of the Stephania cp genomes revealed 5 highly variable regions, including 3 intergenic regions (trnH-psbA, trnD-trnY, trnP) and two protein coding genes (rps16 and ndhA). The identified mutational hotspots of Stephania plants exhibited multiple SNP sites and Gaps, as well as different Ka/Ks ratio values. In addition, five pairs of specific primers targeting the divergence regions were accordingly designed, which could be utilized as potential molecular markers for species identification, population genetic and phylogenetic analysis in Stephania species. Phylogenetic tree analysis based on the conserved chloroplast protein coding genes indicated a sister relationship between S. tetrandra and the monophyletic group of S. japonica and S. kwangsiensis with high support values, suggesting a close genetic relationship within Stephania plants. However, two S. tetrandra vouches from different regions failed to cluster into one clade, confirming the occurrences of genetic diversities and requiring further investigation for geographical tracing strategy. CONCLUSIONS: Overall, we provided comprehensive and detailed information on the complete chloroplast genome and identified nucleotide diversity hotspots of Stephania species. The obtained genetic resource of S. tetrandra from Zhejiang Province would facilitate future studies in DNA barcode, species discrimination, the intraspecific and interspecific variability and the phylogenetic relationships of Stephania plants.

Characterization of the complete chloroplast genome of the medicinal plant Orixa japonica (Rutaceae) in Zhejiang Province and its phylogenetic analysis within family Rutaceae.

Orixa japonica Thunb. is an important medicinal plant belonging to the family Rutaceae. In this study, we determined the the complete chloroplast (cp) genome of O. japonica, which was 158,525 bp in length containing one large single copy region (85,965 bp), one small single copy region (18,552 bp), and a pair of inverted repeat regions (27,004 bp each). A total of 134 genes were annotated in the cp genome, including 88 protein coding genes, 37 tRNA genes, eight rRNA genes, and one pseudo gene ycf1. According to the phylogenetic analysis, O. japonica clustered together with Casimiroa edulis with high bootstrap value, indicating a close genetic relationship with subfamily Amyridoideae.

The complete chloroplast genome of the medicinal plant Polygonum cuspidatum (Polygonaceae) and its phylogenetic implications within the subfamily Polygonoideae.

Polygonum cuspidatum Siebold & Zucc. is a well-known and widely used medical plant to treat arthritis, gout and inflammation. In this study, we determined the complete chloroplast genome sequence of P. cuspidatum from Zhejiang Province. The assembled chloroplast (cp) genome was 163,183 bp in length, containing two inverted repeated (IR) regions of 30,859 bp each, a large single copy (LSC) region of 87,905 bp, and a small single copy (SSC) region of 13,560 bp. The genome encodes 131 genes, consisting of 86 protein-coding, 37 tRNA, and eight rRNA genes. The overall GC content of P. cuspidatum is 37.53%, with the highest GC content of 41.27% in the IR region. The 86 protein-coding genes encode 27,597 amino acids in total, most of which use the initiation codon ATG, except the ndhD gene which starts with ACG. The length of the tRNA genes range from 48 bp to 88 bp, with the highest GC content of 62.16% in tRNA-Arg (ACG) and tRNA-Asp (GUC). A total of 66 simple sequence repeats are identified in the cp of P. cuspidatum. Phylogenetic analysis indicated a sister relationship between P. cuspidatum and Fallopia sachalinensis, suggesting a close genetic relationship between the genera of Polygonum and Fallopia. This work provides basic genetic resources for investigating the evolutionary status and population genetics of this important medicinal species.

Complete chloroplast genome sequence of the medicinal plant ramie (Boehmeria nivea L. gaud) and its phylogenetic relationship to other Urticaceae species.

Ramie (Boehmeria nivea L. Gaud) is a traditional fiber crop and important medicinal plant belonging to the family Urticaceae. In this study, we determine the complete chloroplast genome sequence of B. nivea. The assembled chloroplast genome is 156065 bp in length and shares the conserved quadripartite structure as other cp genomes in Boehmeria. The genome contains 131 genes, including 84 protein genes, 8 rRNA genes, 37 tRNA genes and 2 pseudo genes. There are 17 duplicated genes in the IR region. The overall GC content of B. nivea is 36.33%, with the highest GC content of 42.72% in IR region. A total of 67 simple sequence repeats are identified in the cp genome of B. nivea. Phylogenetic analysis demonstrated that B. nivea clustered together with B. tomentosa, further forming a monophyletic group with the species of Debregeasia and Pipturus. This work provides basic genetic resources for developing robust markers and investigating the population genetics diversities for B. nivea.

Characterization of the complete chloroplast genome of Oxalis corymbosa DC. (Oxalidaceae), a medicinal plant from Zhejiang Province.

Oxalis corymbosa DC. is an important medicinal and edible perennial herb belonging to the wood-sorrel family Oxalidaceae. In this study, we report the complete chloroplast (cp) genome sequence of O. corymbosa. The assembled chloroplast genome was 151,351 bp in length, containing two inverted repeated (IR) regions of 24,587 bp each, a large single copy (LSC) region of 85,476 bp, and a small single copy (SSC) region of 16,701 bp. The genome encodes 128 genes, consisting of 82 protein-coding genes, 37 tRNA genes, eight rRNA genes, and one pseudogene (ycf1). The 82 protein-coding genes encode 25,751 amino acids in total, most of which use the initiation codon ATG, except rps19 and psbC genes start with GTG. The lengths of the tRNA genes range from 71 bp to 93 bp, with the highest GC content of 62.16% in tRNA-Arg (ACG). The overall GC content of O. corymbosa is 36.47%, with the highest GC content of 42.64% in IR region. In addition, a total of 74 simple sequence repeats were identified in the cp genome of O. corymbosa. Phylogenetic analysis indicated a sister relationship between O. corymbosa and O. drummondii, suggesting a close genetic relationship between the two Oxalis species. This work provides basic genetic resources for investigating the evolutionary status and population genetics diversities for this medicinal species.

Molecular structure and phylogenetic analysis of complete chloroplast genomes of medicinal species Paeonia lactiflora from Zhejiang Province.

Paeonia lactiflora is a geo-authentic and superior medicinal plant in Zhejiang province. Here, we report the complete chloroplast genome sequence of P. lactiflora. The total genome size of P. lactiflora is 153,405 bp in length, including a small single-copy (SSC) region of 16,969 bp, a large single-copy (LSC) region of 84,340 bp separated by a pair of inverted repeats (IRs) of 26,048 bp. The overall annotated gene number is 109, containing 76 protein-coding genes, 29 tRNAs and 4 rRNAs. The entire GC content of P. lactiflora is 38.43%, with the highest GC content of 42.99% in IR region. A total of 52 simple sequence repeats are identified in the cp genome of P. lactiflora. Phylogenetic analysis indicated a sister relationship between P. lactiflora and P. veitchii, and supported a unique evolutionary status of Family Paeoniaceae. This work provides a valuable genetic resource to develop robust markers and investigate the population genetics diversities for this famous medicinal species.

[Morphological characteristics identification and molecular DNA barcoding analysis of Hippocampus spinosissimus].

The combination of morphological characteristics and DNA barcodes was used to a systematic study of Hippocampus spinosissimus,laying the foundation for rapid and accurate identification for the medical seahorse species. According to the reported literature and observation on seahorse samples,the typical characteristics of the H. spinosissimus include highly developed spiny,much short nose,single or double cheeks and strongly developed spines bordering pouch. Genomic DNAs of H. spinosissimus and other related seahorse species were extracted using the TIANamp Marine Animals DNA Kit. The COⅠ and ATP6 genes were amplified and sequenced in both directions. After the verification by Blast,the GC content,intraspecific and interspecific genetic distance,and the Neighbor joining( NJ) phylogenetic trees were analyzed by MEGA 7. The lengths of the COⅠ and ATP6 genes were 649 bp and 602-603 bp,respectively,with the average GC content of 39. 96% and 35. 37%. The maximum intraspecific genetic distances in H. spinosissimus based on COⅠ and ATP were both far less than the minimum interspecific genetic distance between H. spinosissimus and other seahorses,suggesting a significant barcoding gap. NJ analysis results of COⅠ and ATP6 exhibited that all H. spinosissimus species clustered together,indicating that the two DNA barcode could identify H. spinosissimus from other seahorses accurately and quickly. In addition,H. spinosissimus shared a close genetic relationship between H. kelloggi according to the NJ tree. Furthermore,there exits three stable subgroup structure of H. spinosissimus,indicating that COⅠ and ATP6 barcodes could be applied the indicator for the geographical ecology research of H. spinosissimus. The results obtained the typical morphological and molecular identification characteristics of H. spinosissimus,which played central roles for the development of species identification. This study provides an important basis data for expanding the medical seahorse resources and ensuring the safety of clinical medicine.

Fangxiao Formula alleviates airway inflammation and remodeling in rats with asthma via suppression of transforming growth factor-ß/Smad3 signaling pathway.

Asthma is a common obstructive airway disease characterized by inflammation and remodeling with a progressive decline in lung function. Fangxiao Formula (FXF) is an herbal medicine that has achieved significant clinical benefits toward asthma patients, but the relevant mechanism has not yet been clarified. The aim of this study was to determine the inhibitory effects of FXF on airway inflammation and remodeling, and investigate the activities of TGF­ß/Smads signaling pathway in the rat asthma model. Rats were sensitized by ovalbumin (OVA) for six weeks to establish the asthma experimental model. OVA-challenged animals were randomly divided into 5 groups and received different concentrations of FXF or dexamethasone. The animals in blank control group received saline only. Lung tissues were collected and analyzed for determining the inflammatory cells infiltration, HE and PAS staining, airway wall thickness and collagen deposition. The productions of inflammatory cytokine productions were analyzed by ELISA in the bronchoalveolar lavage (BAL) fluid. Immunohistochemical analysis was performed to measure the expression of α-SMA and PCNA in lung tissue after the treatment of FXF. The levels of TGF-ß were assessed by both immunohistology and western blotting, and the expression of p-Smad2/3 proteins were determined by western blotting analysis. Our results indicated that FXF attenuated the infiltration of inflammatory cells, decreased the production of Th2 cytokines and simultaneously increased the levels of Th1 cytokine in the asthma rat model. In addition, FXF reduced allergen-induced increased airway wall thickness, goblet cell hyperplasia and collagen deposition. Furthermore, the expression levels of TGF-ß and p-Smad3 were obviously reduced after the treatment of FXF. These results indicate that FXF alleviates airway inflammation and remodeling by restoring the balance of Th1/Th2 cytokines and the TGF-ß/Smad-3 pathway, therefor providing potential therapeutic approach for asthmatic patients.

[Current status and future prospect of pharmacotherapy for pancreatic cancer].

Pancreatic cancer is the most common digestive tract tumor with an increasing incidence in recent years. The poor prognosis of pancreatic cancer is mainly because of the inability of detecting tumor at an early stage,its high potential for early dissemination,and its relatively poor sensitivity to chemotherapy. Most patients have lost the opportunity for surgery when they are diagnosed,which resulted in an urgent need for the development of more effective and safe therapies for pancreatic cancer. However,the current clinical cancer chemotherapy based on gemcitabine leads to poor prognosis in pancreatic patients. With the continuous research on the biological and cellular signaling pathways of pancreatic cancer,there have emerged a great many of novel agents,including new chemotherapeutic,targetable and immune-modulatory drugs,and some drugs have achieved encouraging results. Furthermore,as an alternative and supplementary method,traditional Chinese medicine has shown good application prospects in the field of pancreatic cancer treatment. This article reviews the current status of drug therapy for pancreatic cancer,summarizes the strength and weakness of existing therapeutic drugs in the application process,gives prospects of possible breakthroughs for the pharmacotherapy in the future,and provides certain new ideas and lessons for subsequent drug development.

The complete chloroplast genome sequence and phylogenetic analysis of the medicinal plant Lysimachia hemsleyana.

Lysimachia hemsleyana Maxim. is an important medical plant in the Family Primulaceae. In this study, we determined the complete chloroplast genome of L. hemsleyana. It is 155,618 bp in length, containing a large single copy (LSC) region of 85,615 bp, a small single copy (SSC) region of 17,861 bp, which were separated by a pair of inverted repeat (IR) regions of 26,071bp. The complete chloroplast genome of L. hemsleyana encoded a total of 134 genes, including 89 protein-coding genes with the pseudogene of ycf1, 8 ribosomal RNA genes and 37 transfer RNA genes. Phylogenetic analysis revealed that L. hemsleyana was most closely related to the Korea endemic plant Lysimachia coreana with high bootstrap support value. This work provides basic molecular information that would be useful for further investigation on conservation genetics and evolutionary relationships of L. hemsleyana.

Total Saponins of Rubus Parvifolius L. Exhibited Anti-Leukemia Effect in vivo through STAT3 and eIF4E Signaling Pathways.

OBJECTIVE: To investigate the anti-leukemia effect of total saponins of Rubus parvifolius L. (TSRP) on K562 cell xenografts in nude mice and the mechanisms of action. METHODS: The K562 cell xenografts in nude mice were established, and then randomly divided into 5 groups, the control group, the cytosine arabinoside group(Ara-c) and 3 TSRP groups (20, 40 and 100 mg/kg). The tumor volume and mass of each group of nude mice were measured and the anti-tumor rates of TSRP were calculated subsequently. The apoptosis status of tumor cells was detected by hematoxylin-eosin (HE) and terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining analysis. Finally, the activities of apoptosis related signaling of signal transducer and activator of transcription 3 (STAT3), eukaryotic initiation factor 4E (eIF4E) and B-cell lymphoma-2 (bcl-2) were determined with immunohistochemistry tests. RESULTS: Subcutaneous injection of K562 cells induced tumor formation in nude mice, and the TSRP treated group showed a signifificant inhibitory effect on tumor formation. The nude mice treated with TSRP showed a signifificant decrease in tumor growth rate and tumor weight in comparison to the control group (all P<0.05). The HE staining and TUNEL assay showed that TSRP induced cell death by apoptosis. The immunohistochemical assay showed down-regulation of the bcl-2 gene in the TSRP treated cells. The phosphorylation levels of eIF4E and STAT3 were decreased obviously after the treatment of TSRP. CONCLUSION: TSRP had an excellent tumor-suppressing effect on K562 cells in the nude mice xenograft model, suggesting that TSPR can be developed as a promising anti-chronic myeloide leukemia drug.

[Anti-lung cancer mechanisms of diterpenoid tanshinone via endoplasmic reticulum stress-mediated apoptosis signal pathway].

At present, lung cancer ranks second and first respectively in the incidence and the mortality among malignant tumors. It is urgent to find new effective anti-lung cancer drugs with less side effects and relatively defined mechanisms. Endoplasmic reticulum stress (ERS)-mediated apoptosis pathway is an effective way to promote tumor cell apoptosis; diterpenoid tanshinone (DT), an effective part separated from Salviae Miltiorrhizae Radix et Rhizoma, was found to have an anti-lung cancer effect in previous studies via ERS-induced PERK-EIF2α pathway. In this paper, human lung adenocarcinoma PC9 cell line and nude mouse transplantation tumor model were applied to verify the anti-lung cancer effect of DT in vivo and in vitro, and illuminate the potential mechanism via ERS induced IRE1α/caspase 12 apoptosis pathway. The results showed that in vivo, DT could promote PC9 cell apoptosis in a concentration-dependent manner, up-regulate Bip, IRE1 and TRAF2 protein expressions in tumor tissue, reduce tumor weight and alleviate bodyweight loss. In vitro, DT inhibited the proliferation of PC9 cell line in a concentration-dependent manner, and destroyed the structure of mitochondria in PC9 cell, promoted Bax, IRE1α, Bip, TRAF2 and caspase 12 protein expressions, lower Bcl-2 protein expression in a time-dependent manner. DT shows a good effect on anti-lung cancer both in vivo and in vitro. The mechanism is related to the activation of ERS-induced IRE1α/caspase 12 apoptosis pathway and the promotion of cell apoptosis. ERS-mediated apoptosis pathway may be an important target of DT on anti-lung cancer.

[Progress of regulation of leukemia stem cells of chronic myeloid leukemia by autophagy].

Leukemia stem cells (LSC) that were found in chronic myeloid leukemia (CML) responsible for the abnormal proliferation with the potential of self-renewal and multi-directional differentiation are involved in the pathophysiological process for drug resistance and relapse of CML. Autophagy, a conservative lysosomal degradation process that mediates cell degradation and recycling process, plays crucial roles in maintaining the homeostasis and function of intracellular environment. Recent studies suggested that autophagy is involved in the regulation of LSC differentiation and also closely related to the chemo-sensitivity of CML. In this review, we focused on the role of autophagy on chemotherapy sensitivity of CML as well as the leukemia stem cell function for the development of new anti-leukemia drugs.

[Effect of cryptotanshinone on imatinib sensitivity and P-glycoprotein expression of chronic myeloid leukemia cells].

Cryptotanshinone (CPT), a lipid soluble active compound in Salvia miltiorrhiza, has a significant inhibitory effect on multiple malignant tumors, e. g. chronic myeloid leukemia (CML) cells and can effectively enhance imatinib's chemotherapeutic effect. However, its functional molecular mechanism remained unclear. In this experiment, the authors conducted a systematic study on the effect of CPT on the imatinib sensitivity and P-glycoprotein (P-gp) expression in CML cells by using CML cells K562 and imatinib persister K562-R. The MTT assays were performed to determine CPT's impact on the inhibitory effect of imatinib. Annexin V-FITC/PI staining analysis was used to detect the changes in the cell apoptosis rate. The active changes in apoptosis regulatory proteins Caspase-3, Caspase-9 and PARP were determined by Western blot. After the cells were pretreated with the gradient concentration of CPT, the expression of P-gp was analyzed by Western blot and flow cytometry. The changes in intracellular concentrations of imatinib were determined by HPLC analysis. The results indicated that the pretreatment with CPT significantly increased the proliferation inhibiting and apoptosis inducing effects of imatinib on K562 and K562-R cells as well as the degradation product expression of pro-apoptotic proteins Caspase-3, Caspase-9 and PARP, with a significant difference with the control group (P < 0.01). However, CPT showed no impact on the P-gp expression in CML cells and the intracellular concentrations of imatinib. In summary, the findings suggested that CPT enhanced the sensitivity of CML cells to imatinib. Its mechanism is not dependent on the inhibition in P-gp expression and the increase in intracellular drug concentration.

Cryptotanshinone acts synergistically with imatinib to induce apoptosis of human chronic myeloid leukemia cells.

Imatinib resistance has emerged as a significant clinical problem in chronic myeloid leukemia (CML) treatment. In this study, we investigated the effect and mechanism of combination treatment with imatinib and cryptotanshinone (CPT) in CML cells. Cotreatment with imatinib and CPT showed a significant synergistic killing effect in both imatinib sensitive and resistant CML cell lines, as well as primary CML cells. Furthermore, combination treatment induced apoptosis significantly, as indicated by increases in apoptotic cell fraction and activities of proapoptotic proteins. Subsequent studies revealed that CPT significantly inhibited Bcr/Abl protein expression, as well as phosphorylation expression levels of signal transducer and activator of transcription 3 (STAT3), mammalian target of rapamycin (mTOR) and eukaryotic translation initiation factor 4E (eIF4E), which are critical mediators of Bcr/Abl transformation. Furthermore, CPT in combination with imatinib dramatically decreased the activity of the Bcr/Abl pathway in both K562 and K562-R cells. Our results demonstrated that CPT increased imatinib-induced apoptosis in a Bcr/Abl dependent manner, suggesting a novel strategy for the treatment of CML.

Saponins from Rubus parvifolius L. induce apoptosis in human chronic myeloid leukemia cells through AMPK activation and STAT3 inhibition.

BACKGROUND: Saponins are a major active component for the traditional Chinese medicine, Rubus parvifolius L., which has shown clear antitumor activities. However, the specific effects and mechanisms of saponins of Rubus parvifolius L. (SRP) remain unclear with regard to human chronic myeloid leukemia cells. The aim of this study was to investigate inhibition of proliferation and apoptosis induction effects of SRP in K562 cells and further elucidate its regulatory mechanisms. MATERIALS AND METHODS: K562 cells were treated with different concentrations of SRP and MTT assays were performed to determine cell viability. Apoptosis induction by SRP was determined with FACS and DAPI staining analysis. Western blotting was used to detect expression of apoptosis and survival related genes. Specific inhibitors were added to confirm roles of STAT3 and AMPK pathways in SRP induction of apoptosis. RESULTS: Our results indicated that SRP exhibited obvious inhibitory effects on the growth of K562 cells, and significantly induced apoptosis. Cleavage of pro-apoptotic proteins was dramatically increased after SRP exposure. SRP treatment also increased the activities of AMPK and JNK pathways, and inhibited the phosphorylation expression level of STAT3 in K562 cells. Inhibition of the AMPK pathway blocked the activation of JNK by SRP, indicating that SRP regulated the expression of JNK dependent on the AMPK pathway. Furthermore, inhibition of the latter significantly conferred resistance to SRP pro- apoptotic activity, suggesting involvement of the AMPK pathway in induction of apoptosis. Pretreatment with a STAT3 inhibitor also augmented SRP induced growth inhibition and cell apoptosis, further confirming roles of the STAT3 pathway after SRP treatment. CONCLUSIONS: Our results demonstrated that SRP induce cell apoptosis through AMPK activation and STAT3 inhibition in K562 cells. This suggests the possibility of further developing SRP as an alternative treatment option, or perhaps using it as adjuvant chemotherapeutic agent for chronic myeloid leukemia therapy.

Induction of cytotoxicity and apoptosis in human gastric cancer cell SGC-7901 by isovaltrate acetoxyhydrin isolated from Patrinia heterophylla bunge involves a mitochondrial pathway and G2/M phase cell cycle arrest.

BACKGROUND: Our previous study demonstrated cytotoxicity of a crude extract from Patrinia heterophylla Bunge (PHEB). In the present study, we aimed to investigate the effects of isovaltrate acetoxyhydrin (IA) isolated from PHEB on the gastric cancer cell SGC-7901, in order to explore a potential treatment for gastric cancer. METHODS: MTT assays were employed to determine the effects of IA on cell vitality and proliferation, with monitoring of cell morphology changes and examination of apoptosis with Annexin V-PI staining. Flow cytometry was used to assess cell cycle progression and mitochondrial membrane potential. The activity of caspase 3, 9 was evaluated by spectrophotometry, and the protein levels of Bax, Bcl2 and Cyclin B1 were analyzed with Western blotting of total proteins extracted from cultured cells. RESULTS: The results demonstrated direct toxicity of IA towards SGC-7901 cells. Evidence of apoptosis included blebbing and chromatin condensation. Annexin V-PI assays revealed early apoptosis, involving rapid depolarization of mitochondrial membranes and activity of caspase 3, 9 signaling pathways. Western blotting showed that Bcl2 and Bax proteins was down- and up-regulated, respectively, and cyclin B1 was up-regulated. Cell cycle analysis further indicated that IA could induce G2/M phase arrest in SGC-7901 cells. CONCLUSIONS: In conclusion, we believe that IA induces apoptosis of SGC-7901 cells, therefore providing a potential therapeutic agent for treatment of gastric cancer.