川连抗癌的实验研究

目的通过对动物体内抗癌实验的观察研究,验证中药川连具有抑制和治疗肿瘤的生物学特性。方法选取80只昆明小鼠,于前肢皮下接种小鼠肉瘤细胞。将动物随机分为对照组和川连治疗组。治疗组每日给予川连汤剂稀释液（0．5mg,瘤内注射,1次／d）,连续10d;对照组注射同体积生理盐水。结果连续观察70d,治疗组小鼠存活率100％,对照组小鼠存活率为0％。结论中药川连具有抑制和治疗肿瘤作用,其抗肿瘤作用机制有待于进一步研究。     

Antimicrobial properties of berberines alkaloids in Coptis chinensis Franch by microcalorimetry

The growth thermogenic curves of Escherichia coli (E. coli) affected by berberine, coptisine and palmatine were determined quantitatively by microcalorimetry. The power–time curves of E. coli with and without the three berberines alkaloids (BA) were acquired, meanwhile the extent and duration of inhibitory effects on the metabolism were evaluated by growth rate constant (k), half-inhibitory ratio (IC₅₀), peak time of maximum heat-output power (t_p), total heat-production (Q_t) and so on. The inhibitory effects of BA on E. coli revealed that the sequence of their antimicrobial activity was berberine > coptisine > palmatine. The functional groups methylenedioxy at C2 and C3 on phenyl ring improve antimicrobial activity more remarkably than methoxyl at C2 and C3 on phenyl ring. However, the antimicrobial activity does not vary significantly with methylenedioxy or methoxyl at C9 and C10 on phenyl ring.     

江苏引种黄连副产物的生物碱含量测定

利用薄层扫描法和高效液相色谱法测定了江苏引种黄连根茎以外的副产物（须极、连渣、叶片、叶柄）中生物碱的含量,其中４年生黄连叶片生物碱含量达２．４３％,叶柄达３．７５％,６年一黄连须根生物碱含理达５．６７％,连渣达６．３２％,建议充分利用须根、连渣、叶片、叶柄等副产物。     

Correlation analyses between molecular perspective and phytochemical variations in Coptis chinensis Franch

Coptis chinensis Franch. (Weilian in Chinese) is an important medicinal plant used in traditional Chinese medicines. The identification of habitats associated with good quality plant material is a challenge. In this study, we determined 59 samples from 12 different habits. Other than the samples from Zhenping, the content of six selected alkaloids in C. chinensis did not differ significantly among the habits. Furthermore, the results of the genetic analysis showed that the genetic diversity and the genetic distance among the samples were low, suggesting that the C. chinensis samples from different habits had the same genetic characteristics. These results would suggest that the quality of the drugs are not influenced by the habitats the plant is growing in.     

Antifungal effects of volatile organic compounds from the endophytic fungus Cryptosporiopsis ericae Cc-HG-7 isolated from Coptis chinensis Franch

In this study, 136 strains of endophytic fungi were isolated from the Chinese traditional medicinal plant Coptis chinensis Franch. Of these, 129 strains were classified into 12 different genera according to morphological traits and internal transcribed spacer (ITS) gene sequence analyses. Their antifungal activities were assessed against the following fungi: Magnaporthe oryzae, Pythium graminicola, Cylindrocarpon destructans, Fusarium oxysporum, Cercospora zeae-maydis, Sclerotinia sclerotiorum, Setosphaeria turcica and Botrytis cinerea. Fourteen endophytic strains were active against at least one of the selected fungi. The most active strain Cc-HG-7 identified as Cryptosporiopsis ericae displayed inhibition rates of 81.42% and 72.00%, respectively, against S. sclerotiorum and S. turcica in dual culture technique. The volatile antifungal compounds were identified using headspace solid-phase microextraction, followed by gas chromatography mass spectrometry to investigate the potential biocontrol mechanisms of strain Cc-HG-7. The results suggested that the strain Cc-HG-7 could be a potential agent for the biological control of S. sclerotiorum and S. turcica.     

Differentiating Coptis chinensis from Coptis japonica and other Coptis species used in Coptidis Rhizoma based on partial trnL-F intergenic spacer sequences

Dried rhizomes of Coptis species are utilized as “Coptidis Rhizoma” (CR), an important herbal medicinal material in traditional Chinese medicine. Almost all CRs traded in the Korean herbal medicine market originate from Coptis chinensis (“Chun Hwang-Lyun” in Korean medical terminology). Other minor CRs originate from Coptis japonica (“Il Hwang-Lyun”). Although there is an obvious discrepancy in the price of traded CRs in the herbal market depending on the Coptis species, CRs originating from C. chinensis and C. japonica are often confused. Furthermore, the CR traded as “Chun Hwang-Lyun” is occasionally mixed with rhizomes of Coptis deltoidea and/or Coptis omeiensis. Therefore, we sought to discriminate C. chinensis from C. japonica, as well as C. deltoidea and C. omeiensis, by using nucleotide sequence differences in the partial trnL-F intergenic spacer. We developed an efficient real-time polymerase chain reaction (PCR)-based discrimination assay to separate samples of C. chinensis from those of C. japonica without the need to separate the DNA markers by using gel electrophoresis. In addition, we developed a multiplex PCR method with which we were able to discriminate samples of C. chinensis from those of C. deltoidea and C. omeiensis by amplifying the 153-bp DNA marker in C. chinensis in a single PCR process.     

Enhanced inhibition of prostate cancer xenograft tumor growth by combining quercetin and green tea

The chemopreventive activity of green tea (GT) is limited by the low bioavailability and extensive methylation of GT polyphenols (GTPs) in vivo. We determined whether a methylation inhibitor quercetin (Q) will enhance the chemoprevention of prostate cancer in vivo. Androgen-sensitive LAPC-4 prostate cancer cells were injected subcutaneously into severe combined immunodeficiency (SCID) mice one week before the intervention. The concentration of GTPs in brewed tea administered as drinking water was 0.07% and Q was supplemented in diet at 0.2% or 0.4%. After 6-weeks of intervention tumor growth was inhibited by 3% (0.2% Q), 15% (0.4% Q), 21% (GT), 28% (GT+0.2% Q) and 45% (GT+0.4% Q) compared to control. The concentration of non-methylated GTPs was significantly increased in tumor tissue with GT+0.4% Q treatment compared to GT alone, and was associated with a decreased protein expression of catechol-O-methyltransferase and multidrug resistance-associated protein (MRP)-1. The combination treatment was also associated with a significant increase in the inhibition of proliferation, androgen receptor and phosphatidylinositol 3-kinase/Akt signaling, and stimulation of apoptosis. The combined effect of GT+0.4% Q on tumor inhibition was further confirmed in another experiment where the intervention started prior to tumor inoculation. These results provide a novel regimen by combining GT and Q to improve chemoprevention in a non-toxic manner and warrant future studies in humans.     

Midazolam induces cellular apoptosis in human cancer cells and inhibits tumor growth in xenograft mice

Midazolam is a widely used anesthetic of the benzodiazepine class that has shown cytotoxicity and apoptosisinducing activity in neuronal cells and lymphocytes. This study aims to evaluate the effect of midazolam on growth of K562 human leukemia cells and HT29 colon cancer cells. The in vivo effect of midazolam was investigated in BALB/c-nu mice bearing K562 and HT29 cells human tumor xenografts. The results show that midazolam decreased the viability of K562 and HT29 cells by inducing apoptosis and S phase cell-cycle arrest in a concentration-dependent manner. Midazolam activated caspase-9, capspase-3 and PARP indicating induction of the mitochondrial intrinsic pathway of apoptosis. Midazolam lowered mitochondrial membrane potential and increased apoptotic DNA fragmentation. Midazolam showed reactive oxygen species (ROS) scavenging activity through inhibition of NADPH oxidase 2 (Nox2) enzyme activity in K562 cells. Midazolam caused inhibition of pERK1/2 signaling which led to inhibition of the anti-apoptotic proteins Bcl-X_L and XIAP and phosphorylation activation of the pro-apoptotic protein Bid. Midazolam inhibited growth of HT29 tumors in xenograft mice. Collectively our results demonstrate that midazolam caused growth inhibition of cancer cells via activation of the mitochondrial intrinsic pathway of apoptosis and inhibited HT29 tumor growth in xenograft mice. The mechanism underlying these effects of midazolam might be suppression of ROS production leading to modulation of apoptosis and growth regulatory proteins. These findings present possible clinical implications of midazolam as an anesthetic to relieve pain during in vivo anticancer drug delivery and to enhance anticancer efficacy through its ROS-scavenging and pro-apoptotic properties.     

Induction of apoptosis in prostate tumor PC-3 cells and inhibition of xenograft prostate tumor growth by the vanilloid capsaicin

Capsaicin, the pungent ingredient of hot chilli pepper, has been recently shown to induce apoptosis in several cell lines through a not well known mechanism. Here, we investigated the role of the vanilloid capsaicin in the death regulation of the human cancer androgen-resistant cell line PC-3. Capsaicin inhibited the growth of PC-3 with an IC₅₀ of 20 μM cells and induced cell apoptosis, as assessed by flow cytometry and nuclei staining with DAPI. Capsaicin induced apoptosis in prostate cells by a mechanism involving reactive oxygen species generation, dissipation of the mitochondrial inner transmembrane potential (ΔΨ_m) and activation of caspase 3. Capsaicin-induced apoptosis was not reduced by the antagonist capsazepine in a dose range from 0.1 μM to 20 μM, suggesting a receptor-independent mechanism. To study the in vivo effects of capsaicinoids, PC-3 cells were grown as xenografts in nude mice. Subcutaneous injection of either capsaicin or capsazepine (5 mg/kg body weight) in nude mice suppressed PC-3 tumor growth in all tumors investigated and induced apoptosis of tumor cells. Our data show a role for capsaicin against androgen-independent prostate cancer cells in vitro and in vivo and suggest that capsaicin is a promising anti-tumor agent in hormone-refractory prostate cancer, which shows resistance to many chemotherapeutic agents.     

黄连细胞培养物中小檗碱的分离与鉴定

用黄连幼叶切块,接种在含1．0～2．0mg／L 2,4-D和0．1mg／LKT的6．7-v固体培养基上,诱导形成愈伤组织。选择较松散的愈伤组织转入液体悬浮培养,获得游离细胞和细胞聚集体。通过平板培养和细胞株的筛选．选择小檗碱含量较高的细胞株。经35次转代培养后,进行固体、液体培养。收集悬浮培养细胞进行化学提取和分离,获得黄色晶体,经TLc、UV、IR、MS鉴定分析为小檗碱,与天然植物提取分离的小檗碱具有相同的生理活性。     

黄连ISSR反应条件优化的研究

以黄连(味连,Coptis Chinensis Franch.)基因组DNA为模板,通过单因子、双因子实验研究了ISSR反应体系中主要成分（Mg²⁺、dNTP、引物、模板、Taq DNA聚合酶）以及热循环参数（退火温度、循环数、变性时间、退火时间、延伸时间）对扩增结果的影响,并找出各自的最适条件,建立了适合黄连ISSR分析的反应体系和扩增程序,即在25μL反应体系中,内含1×PCR buffer、1.5mmol·L^-1 Mg²⁺、200μmol·L^-1 dNTP、0.3 μmol·L^-1引物、40 ng模板、1 U TaqDNA聚合酶。扩增程序为94℃预变性5 min,然后进行35个循环：94℃变性30 s,（据不同引物的退火温度）复性1 min,72℃延伸1.5 min,循环结束后72℃延伸7 min,-4℃保存。这一优化系统的建立为今后利用ISSR标记技术进行黄连鉴定及种质遗传多样性分析提供了一个标准化程序。     

Jolkinolide B induces cell cycle arrest and apoptosis in MKN45 gastric cancer cells and inhibits xenograft tumor growth in vivo

Gastric cancer is one of the most common digestive carcinomas throughout the world and represents high mortality. There is an urgent quest for seeking a novel and efficient antigastric cancer drug. Euphorbia fischeriana Steud had long been used as a traditional Chinese medicine for the treatment of cancer. According to the basic theory of traditional Chinese medicine, its antitumor mechanism is ‘to combat poison with poison’. However, its effective material foundation of it is still ambiguous. In our previous work, we studied the chemical constituents of E. fischeriana Steud. Jolkinolide B (JB) is an ent-abietane-type diterpenoid we isolated from it. The purpose of the present study was to investigate the antigastric effect and mechanism of JB. Results revealed that JB could suppress the proliferation of MKN45 cells in vitro and inhibit MKN45 xenograft tumor growth in nude mice in vivo. We further investigated its anticancer mechanism. On the one hand, JB caused DNA damage in gastric cancer MKN45 cells and induced the S cycle arrest by activating the ATR-CHK1-CDC25A-Cdk2 signaling pathway, On the other hand, JB induced MKN45 cells apoptosis through the mitochondrial pathway, and ultimately effectively inhibited the growth of gastric cancer cells. These results suggest that JB appears to be a promising candidate drug with antigastric cancer activity and warrants further research.     

Persistent pain accelerates xenograft tumor growth of breast cancer in rat

Pain occurs at all stages of the patients who suffer from cancer. Owing to surgery and bone metastasis, breast cancer patients were usually disturbed by persistent pain. However, the pain-relief-right has not been respected enough in clinical cancer treatment. Whether pain has any adverse effects on cancer development is still unclear. In order to uncover this question, we established two preclinical animal models to explore the effects of pain on the tumor. For the first model, we mimicked neuropathic pain by sciatic nerve ligation on rats with xenograft tumor subcutaneously. For the second model, we mimicked the bone cancer pain by injecting tumor cell suspension into the tibial medullary cavity of rats with xenograft tumor subcutaneously. The rats with persistent pain showed higher tumor volume and tumor weight compared with the group without pain. Interestingly, when the neuropathic pain and bone cancer pain were relieved by drug administration, both the tumor volume and tumor weight were lowered compared with the group without pain relief. In summary, our study indicated that persistent pain acted as a contributing factor to tumor growth. Moreover, the pain relief could weakened the accelerating role of pain in tumor growth. Thus, we should be paid more attention to the cancer patients with persistent pain as well as cancer treatment.     

Inhibition of xenograft tumor growth in mice by gold nanoparticle-assisted delivery of short hairpin RNAs against Mcl-1L

A prerequisite for the therapeutic use of small RNAs is the development of a method that can deliver them into animals. Previous studies have shown the capability of functionalized gold nanoparticles to serve as a general platform for loading and delivering DNA oligonucleotides and short hairpin RNAs (shRNAs) into cultured human cells. Here, we report the ability of the gold nanoparticle-assisted gene delivery system (AuNP-GDS) to deliver shRNA to a xenograft tumor in a mouse model. AuNP-GDS delivery of in vitro synthesized shRNA targeted to the Mcl-1L gene knocked down levels of Mcl-1L mRNA and protein by ∼36% and ∼26%, respectively, which were sufficient to induce apoptosis of the xenograft tumor cells and consequently inhibited the development of the tumor. We demonstrated that our lego-like AuNP-GDS, which can easily load and deliver shRNAs targeted to any gene of interest into living systems, can deliver shRNAs into xenograft tumors, leading to antitumor activity in an animal model.     

Effect of interferon on the inhibition of tumor cell growth by human peripheral leukocytes

Human leukocyte interferon (HL-IF) enhanced the growth inhibition of tumor cells by the human peripheral leukocytes. There was a dose relation between the enhancement of the growth inhibition of tumor cells and the antiviral activity of interferon. When the ratio of lymphocyte to tumor cell was 10:1 or 50:1, it was recognized that HL-IF enhanced the growth inhibition of tumor cells by lymphocyte. The heterologous IFs--mouse and rabbit IFs--or heat-inactivated or trypsinized IF did not enhance the growth inhibition of tumor cells by lymphocytes. RNase treatment did not reduce the antiviral activity and the growth inhibition.     

Lymphokine-activated tumor inhibition in vivo. I. The local administration of interleukin 2 triggers nonreactive lymphocytes from tumor-bearing mice to inhibit tumor growth

CE-2 is a chemically induced tumor of low immunogenicity in syngeneic BALB/c mice. Nylon wool columns eluting lymphocytes from the spleen of mice bearing clinically evident (5-mm mean diameter) CE-2 tumors (CE-2 TB lymphocytes) do not react with CE-2 cells in vitro, nor are they able to affect their growth in vivo in a Winn-type neutralization assay at 5:1 lymphocyte:tumor cell ratio. However, they become able to inhibit CE-2 tumor growth when 20 U of interleukin 2 (IL 2) in 0.4 ml are injected daily for 10 days at the challenge site. In contrast, mice injected with CE-2 cells and IL 2 only display tumor takes and growth that are not significantly different from those in controls challenged with CE-2 cells alone. This lymphokine-activated tumor inhibition (LATI) is not a peculiarity of the CE-2 tumor-host combination, because different tumors can be inhibited in this way and various TB lymphocytes can initiate it. In these experiments, IL 2-rich 25,000 to 30,000 m.w. fractions were obtained routinely from the culture supernatants of a clone of EL-4 thymoma stimulated with phorbol myristic acetate. Equally active IL 2-rich preparations were obtained from rat spleen cells stimulated with concanavalin A, or from MLA 144 gibbon lymphosarcoma spontaneously releasing IL 2. Treatment of CE-2 TB lymphocytes with various antibody and C, with 2000 rad gamma-irradiation, or fractionation on Percoll density gradients suggested that radioresistant functions of Thy-1.2+, Lyt-1.2+, Lyt-2.2- and of asialo GM1+ cells are independently involved in LATI induction. These lymphocytes inhibit tumor growth by recruiting the radiosensitive effector mechanisms of the recipient mice required for ultimate tumor destruction. CE-2 tumor inhibition by LATI leaves a specific delayed-type hypersensitivity and an immunologic memory, resulting in rejection of a second lethal CE-2 challenge in a significant number of mice.     

An oncolytic adenovirus delivering TSLC1 inhibits Wnt signaling pathway and tumor growth in SMMC-7721 xenograft mice model

Tumor suppressor in lung cancer-1(TSLC1)was first identified as a tumor suppressor for lung cancer,and frequently downregulated in various types of cancers incl...