Antitumor activity and transcriptome sequencing (RNA-seq) analyses of hepatocellular carcinoma cells in response to exposure triterpene-nucleoside conjugates.

Fifteen betulonic/betulinic acid conjugated with nucleoside derivatives were synthesized to enhance antitumor potency and water solubility. Among these, the methylated betulonic acid-azidothymidine compound (8c) exhibited a broad-spectrum of antitumor activity against three tested tumor cell lines, including SMMC-7721 (IC50 = 5.02 µM), KYSE-150 (IC50 = 5.68 µM), and SW620 (IC50 = 4.61 µM) and along with lower toxicity (TC50 > 100 µM) estimated by zebrafish embryos assay. Compared to betulinic acid (<0.05 µg/mL), compound 8c showed approximately 40-fold higher water solubility (1.98 µg/mL). In SMMC-7721 cells, compound 8c induced autophagy and apoptosis as its concentration increased. Transcriptomic sequencing analysis was used to understand the potential impacts of the underlying mechanism of 8c on SMMC-7721 cells. Transcriptomic studies indicated that compound 8c could activate autophagy by inhibiting the PI3K/AKT pathway in SMMC-7721 cells. Furthermore, in the xenograft mice study, compound 8c significantly slowed down the tumor growth, as potent as paclitaxel treated group. In conclusion, methylated betulonic acid-azidothymidine compound (8c) not only increases water solubility, but also enhances the potency against hepatocellular carcinoma cells by inducing autophagy and apoptosis, and suppressing the PI3K/Akt/mTOR signaling pathway.

Killing tumor-associated bacteria with a liposomal antibiotic generates neoantigens that induce anti-tumor immune responses.

Increasing evidence implicates the tumor microbiota as a factor that can influence cancer progression. In patients with colorectal cancer (CRC), we found that pre-resection antibiotics targeting anaerobic bacteria substantially improved disease-free survival by 25.5%. For mouse studies, we designed an antibiotic silver-tinidazole complex encapsulated in liposomes (LipoAgTNZ) to eliminate tumor-associated bacteria in the primary tumor and liver metastases without causing gut microbiome dysbiosis. Mouse CRC models colonized by tumor-promoting bacteria (Fusobacterium nucleatum spp.) or probiotics (Escherichia coli Nissle spp.) responded to LipoAgTNZ therapy, which enabled more than 70% long-term survival in two F. nucleatum-infected CRC models. The antibiotic treatment generated microbial neoantigens that elicited anti-tumor CD8+ T cells. Heterologous and homologous bacterial epitopes contributed to the immunogenicity, priming T cells to recognize both infected and uninfected tumors. Our strategy targets tumor-associated bacteria to elicit anti-tumoral immunity, paving the way for microbiome-immunotherapy interventions.

Synthesis and Biological Evaluation of Novel Allobetulon/Allobetulin-Nucleoside Conjugates as AntitumorAgents.

Allobetulin is structurally similar tobetulinic acid, inducing the apoptosis of cancer cells with low toxicity. However, both of them exhibited weak antiproliferation against several tumor cell lines. Therefore, the new series of allobetulon/allobetulin-nucleoside conjugates 9a-10i were designed and synthesized for potency improvement. Compounds 9b, 9e, 10a, and 10d showed promising antiproliferative activity toward six tested cell lines, compared to zidovudine, cisplatin, and oxaliplatin based on their antitumor activity results. Among them, compound 10d exhibited much more potent antiproliferative activity against SMMC-7721, HepG2, MNK-45, SW620, and A549 human cancer cell lines than cisplatin and oxaliplatin. In the preliminary study for the mechanism of action, compound 10d induced cell apoptosis and autophagy in SMMC cells, resulting in antiproliferation and G0/G1 cell cycle arrest by regulating protein expression levels of Bax, Bcl-2, and LC3. Consequently, the nucleoside-conjugated allobetulin (10d) evidenced that nucleoside substitution was a viable strategy to improve allobetulin/allobetulon's antitumor activity based on our present study.

Impact of Irradiation on the Pharmacokinetics and Biotransformation of Tamoxifen.

BACKGROUND: The optimal procedure for combining radiotherapy (RT) with tamoxifen treatment is controversial as RT may alter the pharmacokinetics and biotransformation of tamoxifen. The present study investigated this potential interaction by assessing the pharmacokinetics of tamoxifen during concurrent and sequential RT. METHOD: Plasma tamoxifen concentration was measured in rats with or without RT 2.0 Gy (RT2.0Gy) or 0.5 Gy (RT0.5Gy) with ultra-high-performance liquid chromatography-tandem mass spectrometry after tamoxifen administration (10 mg/kg, p.o., n = 6). Tamoxifen was either administered 1 h after RT (concurrent condition) or 24 h after RT (sequential condition). RESULTS: Pharmacokinetic data analysis demonstrated that the area under the curve (AUC) and half-life of tamoxifen were 2,004 ± 241 h ng/ml and 6.23 ± 1.21 h, respectively, after tamoxifen administration (10 mg/kg, p.o.). The respective conversion rate of 4-hydroxytamoxifen, N-desmethytamoxifen, and endoxifen for tamoxifen metabolism was 20%, 16%, and 5%. The AUC value of tamoxifen in the RT0.5Gy group was 1.5- to 1.7-fold higher than in the sham and RT2.0Gy groups. The relative bioavailability of tamoxifen at concurrent RT0.5Gy and RT2.0Gy groups ranged from 127% to 202% and from 71% to 152%, respectively. The magnitude of endoxifen, which converted from 4-hydroxytamoxifen and N-desmethyltamoxifen, increased 3- to 5-fold in the concurrent RT groups. By contrast, the AUC of tamoxifen decreased by roughly 24% in the sequential RT2.0Gy group. The conversion ratio of endoxifen was four times higher than that in the sequential RT2.0Gy group compared with rats not exposed to RT. CONCLUSION: The current study provides advanced pharmacokinetic data to confirm the interaction between RT and hormone therapy. Our findings indicate that RT facilitates the metabolism of tamoxifen to active metabolites and thus imply that combination RT-tamoxifen has potential benefits for the treatment of hormone-dependent breast cancer.

Dose-dependent effects of Hedyotis diffusa extract on the pharmacokinetics of tamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen.

Tamoxifen, a widely prescribed medication in premenopausal women diagnosed with hormone-dependent breast cancer, is potentially co-prescribed with Hedyotis diffusa (H. diffusa), particularly in Taiwan. However, no related report has investigated the drug-herb interaction of H. diffusa on the pharmacokinetics of tamoxifen and its metabolites. In the present study, male Sprague-Dawley rats were administered different doses of H. diffusa extract for 5 consecutive days prior to the administration of tamoxifen (10 mg/kg). A validated ultra-liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system was developed to monitor tamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen, and endoxifen in rat plasma. Pharmacokinetic results demonstrated that the area under curves (AUCs) of tamoxifen and the relative bioavailability (%) of tamoxifen were dose-dependently decreased (31-68%) by pre-treatment with H. diffusa extract (3 g/kg and 6 g/kg). In addition, the conversion ratio of 4-hydroxytamoxifen was downregulated (0.5-fold change) and the N-desmethyltamoxifen conversion ratio was upregulated (2-fold change) by high-dose H. diffusa extract. As a result, the relative bioavailability and biotransformation changes affect the clinical efficacy of tamoxifen treatment. These preclinical findings reveal a hitherto unreported interaction between tamoxifen and H. diffusa extract that has implications for their therapeutic efficacy in treating breast cancer.

New phorbol ester derivatives as potent anti-HIV agents.

Tigliane esters show many biological activities, including anti-HIV-1 activity. Our aim in this study was to establish structure-anti-HIV activity relationships for four series of tigliane-type diterpenoids. We synthesized and evaluated 29 new phorbol ester derivatives for anti-HIV activity and for cytotoxicity against human tumor cell lines. Among them, three derivatives, two phorbol-13-monoesters (5d and 5e) and a phorbol-12,13-diester (6a), showed significant anti-HIV activity. We found that better anti-HIV activity was often associated with a shorter acyl ester at C-13. Particularly, compounds with a phenyl ring in the ester side chain exhibited excellent anti-HIV activity and had good safety indexes. Due to its significant anti-HIV potency with a high selectivity index, phorbol-12,13-dicinnamoate (6a) was chosen as the potential candidate for further preclinical trials.

Preclinical Pharmacokinetic Interaction and Histopathological Analyses of Hedyotis diffusa on Sorafenib in Rats.

Sorafenib is one of the most effective target therapeutic agents for patients with late-stage hepatocellular carcinoma. To seek possible alternative adjuvant agents to enhance the efficacy and improve the side effect of sorafenib, Hedyotis diffusa, one of the most prescribed phytomedicines for treating liver cancer patients in Taiwan, was evaluated in this work. We hypothesized that H. diffusa extract is a safety herb combination on the pharmacokinetic and pharmacodynamic effects of sorafenib. We designed treatments of sorafenib in combination with or without H. diffusa extract to examine its pharmacokinetic properties and effects on liver inflammation. The HPLC-photodiode-array method was designed for monitoring the plasma level and pharmacokinetic parameter of sorafenib in rat plasma. The pharmacokinetic results demonstrated that the area under the curve of sorafenib (10 mg/kg, p.o.) in combination with various doses of H. diffusa formulation (1, 3, and 10 g/kg, p.o.) for 5 consecutive days were 5560 ± 1392, 7965 ± 2055, 7271 ± 1371, and 8821 ± 1705 min µg/mL, respectively, no significant difference when compared with sorafenib treatment alone. Furthermore, the hepatic activity in rats administered with sorafenib with/without H. diffusa extract was quantitatively scored by modified hepatic activity index grading. H. diffusa extract in the range of 1 to 10 g/kg per day did not elicit significant herb-induced hepatotoxicity in rats, based on the histopathological study. Consequently, our findings provided positive safety outcomes for the administration of sorafenib in combination with the phytomedicine H. diffusa.

Synthesis and in vitro anticancer activities of biotinylated derivatives of glaucocalyxin A and oridonin.

Fourteen glaucocalyxin A biotinylated derivatives, one glaucocalyxin C biotinylated derivative, and two oridonin biotinylated derivatives were designed and synthesized. Their structures were confirmed from 1H NMR, 13C NMR and HRMS data. The derivatives were evaluated for cytotoxic activities against lung (A549), cervical cancer cell line HeLa derivative (KB), multidrug-resistant KB subline (KB-VIN), triple-negative breast (MDA-MB-231), and estrogen receptor-positive breast (MCF-7) cancer cell lines.[Formula: see text].

Diosgenin Derivatives as Potential Antitumor Agents: Synthesis, Cytotoxicity, and Mechanism of Action.

Thirty-two new diosgenin derivatives were designed, synthesized, and evaluated for their cytotoxic activities in three human cancer cell lines (A549, MCF-7, and HepG2) and normal human liver cells (L02) using an MTT assay in vitro. Most compounds, especially 8, 18, 26, and 30, were more potent when compared with diosgenin. The structure-activity relationship results suggested that the presence of a succinic acid or glutaric acid linker, a piperazinyl amide terminus, and lipophilic cations are all beneficial for promoting cytotoxic activity. Notably, compound 8 displayed excellent cytotoxic activity against HepG2 cells (IC50 = 1.9 µM) and showed relatively low toxicity against L02 cells (IC50 = 18.6 µM), showing some selectivity between normal and tumor cells. Studies on its cellular mechanism of action showed that compound 8 induces G0/G1 cell cycle arrest and apoptosis in HepG2 cells. Predictive studies indicated that p38α mitogen-activated protein kinase (MAPK) is the optimum target of 8 based on its 3D molecular similarity, and docking studies showed that compound 8 fits well into the active site of p38α-MAPK and forms relatively strong interactions with the surrounding amino acid residues. Accordingly, compound 8 may be used as a promising lead compound for the development of new antitumor agents.

Cucurbitane-Type Triterpenoids from the Vines of Momordica charantia and Their Anti-inflammatory Activities.

Seven new cucurbitane-type triterpenoids, kuguaovins A-G (1-7), and five known ones were isolated from the rattans of wild Momordica charantia. Their structures were established by spectroscopic data analyses, including 1D and 2D NMR, IR, and MS techniques. The absolute configurations of the cucurbitanes were determined from NOESY data and partially by X-ray crystallographic analysis. In pharmacological studies, compounds 1-7 and 9-12 exhibited weak anti-inflammatory effects (IC50 = 15-35 µM), based on an anti-NO production assay.

Nanocarrier-mediated immunogenic chemotherapy for triple negative breast cancer.

Triple negative breast cancer (TNBC) does not respond to checkpoint blockade immunotherapy as a result of immunosuppressive tumor microenvironment. To remodel the tumor microenvironment, we developed a liposome formulation to deliver a potential immunogenic cell death (ICD) inducing agent, 17-(allylamino)-17-demethoxygeldanamycin (17-AAG, or tanespimycin), in a tumor targeted manner to reverse the immunosuppressive microenvironment and facilitate the checkpoint blockade immunotherapy. The 17-AAG liposomes was prepared by thin film dispersion methods. The orthotopic 4T1 murine triple negative breast cancer model was studied. 17-AAG delivered by liposome remodeled the immunosuppressive microenvironment, significantly increased tumor infiltrating T cells, lowered the hypoxia level, decreased the suppressive lymphocytes such as tumor associated macrophages and myeloid derived suppressor cells in the tumor microenvironment. In addition, real-time PCR analysis revealed that chemokines and cytokines with immunosuppressive properties were notably reduced, which further facilitated the T cell mediated immunotherapy. Despite the fact that low dose 17-AAG liposomes demonstrated a limited therapeutic effect alone on 4T1 tumor, promising efficacy was observed when 17-AAG liposomes combined with checkpoint blockade immunotherapy. Taken together, 17-AAG liposomes could remodel the immunosuppressive microenvironment of triple negative breast cancer and facilitate the checkpoint blockade immunotherapy.

Kalshiolin A, new lignan from Kalimeris shimadai.

The Asian plant Kalimeris shimadai has been used as food and ethnologic medicine for over a thousand years. In this study, we isolated and identified one new lignan, kalshiolin A (1), and 12 known lignans (2-13). The structures were characterized by the comprehensive analyses of spectroscopic data (HR-ESI-MS, IR, 1D, and 2D-NMR) and the absolute configuration of 1 was determined from ECD calculations. The new compound 1 was also screened for cytotoxic activity but did not show significant potency (IC50 35.9-43.3 µM) against A549, MDA-MB-231, MCF7, KB, and KB-VIN cell lines.

Kalshinoids A-F, Anti-inflammatory Sesquiterpenes from Kalimeris shimadae.

In a study of the potential anti-inflammatory constituents from Kalimeris shimadae, six new sesquiterpenes, kalshinoids A-F (1-6), together with 21 known compounds (7-27), were isolated. The structures and absolute configurations of the new compounds were discerned from extensive spectroscopic analysis, and the absolute configurations of kalshinoids A, B, E, and F were established by ECD calculations. Furthermore, the identified compounds were tested for anti-inflammatory activity as assessed by inhibition of tumor necrosis factor-alpha (TNF-α) in THP-1 cells. Three sesquiterpenes [kalshinoid F, 4(15)-eudesmen-1ß,7,11-triol, and 4α,10α,11-trihydroxy-1ßH,5ßH-guai-7(8)-ene] reduced levels of TNF-α in lipopolysaccharide-stimulated THP-1 cells in a concentration-dependent manner and were more potent than dexamethasone. These natural sesquiterpenes merit further investigation as possible anti-inflammatory agents.

Discovery of an Oleanolic Acid/Hederagenin-Nitric Oxide Donor Hybrid as an EGFR Tyrosine Kinase Inhibitor for Non-Small-Cell Lung Cancer.

Natural triterpenoids, such as oleanolic acid (OA) and hederagenin, display anti-lung cancer effects, and nitric oxide (NO) is associated with some oncogenic signaling pathways. Accordingly, 17 OA/hederagenin-NO donor hybrids were designed, synthesized, and evaluated against tumor cells. The most potent compound, 13, significantly inhibited the proliferation of five tumor cell lines (IC50 4.6-5.2 µM), while hederagenin inhibited the growth of only A549 tumor cells (IC50 > 10 µM). Furthermore, compound 13 showed stronger inhibitory effects on EGFR-LTC kinase activity (IC50 0.01 µM) than hederagenin (IC50 > 20 µM) and inhibited the proliferation of gefitinib-resistant H1975 (IC50 8.1 µM) and osimertinib-resistant H1975-LTC (IC50 7.6 µM) non-small-cell lung cancer (NSCLC) cells. Moreover, compound 13 produced the most NO in H1975 tumor cells, which indicated that NO may play a synergistic role. Collectively, compound 13, a novel hederagenin-NO donor hybrid with a different chemical structure from those of the current FDA-approved EGFR-targeted anti-NSCLC drugs, may be a promising lead compound for the treatment of NSCLC expressing gefitinib-resistant EGFR with a T790 M mutation or osimertinib-resistant EGFR-LTC with an L858R/T790M/C797S mutation. This work should shed light on the discovery of new anti-NSCLC drugs targeting EGFR from natural products.

New Dammarane-type Saponins from Gynostemma pentaphyllum.

Six new dammarane-type saponins, gypenosides CP1-6 (16), along with 19 known compounds 7⁻25, were isolated and characterized from the aerial parts of Gynostemma pentaphyllum. Among these compounds, eight dammarane-type saponins, 2, 5, 6, 7, 11, 12, 13, and 15, exhibited the greatest antiproliferative effects against two human tumor cell lines (A549 and HepG2).

Tenulin and isotenulin inhibit P-glycoprotein function and overcome multidrug resistance in cancer cells.

BACKGROUND: Multidrug resistance (MDR) in cancer is one of the main obstacles in treatment with chemotherapy. Drug efflux through P-glycoprotein is the major mechanism involved in MDR. A potential strategy to provide the best possible clinical outcomes is to develop P-glycoprotein (P-gp) inhibitors from natural products. PURPOSE: The present study investigated the effects of the natural sesquiterpene lactone tenulin and its derivative isotenulin on human P-gp; the mechanisms of kinetic interactions were also explored. METHODS: The human P-gp (ABCB1/Flp-In™-293) stable expression cells were established by using the Flp-In™ system. The effects of tenulin and isotenulin on cell viability were evaluated by SRB assays in established cell lines, sensitive cancer cell line (HeLaS3), and resistant cancer cell line (KB-vin). The transporter inhibition ability was evaluated by calcein-AM uptake assays. The P-gp inhibition kinetics of tenulin and isotenulin were evaluated by rhodamine123 and doxorubicin efflux assays. The ATPase activity was evaluated with the Pgp-Glo™ Assay System. RESULTS: Tenulin and isotenulin significantly inhibited the P-gp efflux function by stimulating P-gp ATPase activity. Tenulin and isotenulin interacted with the effluxes of rhodamine 123 and doxorubicin through a competitive and noncompetitive mechanism, respectively. The combinations of tenulin and isotenulin with chemotherapeutic drugs significantly resensitized MDR cancer cells. CONCLUSION: These results suggested that tenulin and isotenulin are potential candidates to be developed for synergistic treatment of MDR cancers.

Hysteroscopic Management of Retained Products of Conception.

We report a case with retained products of conception (RPOC) managed by hysteroscopic resection. A 45-year-old woman, G5P3SA1AA1, experienced spontaneous abortion on March 8, 2017, and had persistent vaginal bleeding since then. On May 12, 2017, she came to emergency room where endometrium biopsy was done and revealed degenerative gestational tissue with acute inflammation. On May 23, 2017, she followed up at outpatient department where ultrasonography showed no obvious intrauterine abnormalities with endometrial thickness of 6.5 mm. Office hysteroscopy was arranged and RPOC at the posterior uterine wall was suspected. She received hysteroscopic transcervical resection (TCR) of RPOC on May 26, 2017. After TCR, the vaginal bleeding discontinued. The pathology showed degenerative gestational products with acute inflammation. In conclusion, hysteroscopic TCR might be safe and feasible for RPOC.

Concurrent administration of anticancer chemotherapy drug and herbal medicine on the perspective of pharmacokinetics.

With an increasing number of cancer patients seeking an improved quality of life, complementary and alternative therapies are becoming more common ways to achieve such improvements. The potential risks of concurrent administration are serious and must be addressed. However, comprehensive evidence for the risks and benefits of combining anticancer drugs with traditional herbs is rare. Pharmacokinetic investigations are an efficient way to understand the influence of concomitant remedies. Therefore, this study aimed to collect the results of pharmacokinetic studies relating to the concurrent use of cancer chemotherapy and complementary and alternative therapies. According to the National Health Insurance (NHI) database in Taiwan and several publications, the three most commonly prescribed formulations for cancer patients are Xiang-Sha-Liu-Jun-Zi-Tang, Jia-Wei-Xiao-Yao-San and Bu-Zhong-Yi-Qi-Tang. The three most commonly prescribed single herbs for cancer patients are Hedyotis diffusa, Scutellaria barbata, and Astragalus membranaceus. Few studies have discussed herb-drug interactions involving these herbs from a pharmacokinetics perspective. Here, we reviewed Jia-Wei-Xiao-Yao-San, Long-Dan-Xie-Gan-Tang, Curcuma longa and milk thistle to provide information based on pharmacokinetic evidence for healthcare professionals to use in educating patients about the risks of the concomitant use of various remedies.

Pharmacokinetics of Rhodamine 110 and Its Organ Distribution in Rats.

Rhodamine dyes have been banned as food additives due to their potential tumorigenicity. Rhodamine 110 is illegal as a food additive, although its pharmacokinetics have not been characterized, and no accurate bioanalytical methods are available to quantify rhodamine 110. The aim of this study was to develop and validate a fast, stable, and sensitive method to quantify rhodamine 110 using high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) to assess its pharmacokinetics and organ distribution in awake rats. Rhodamine 110 exhibited linear pharmacokinetics and slow elimination after oral administration. Furthermore, its oral bioavailability was approximately 34-35%. The distribution in the liver and kidney suggests that these organs are primarily responsible for rhodamine 110 metabolism and elimination. Our investigation describes the pharmacokinetics and a quantification method for rhodamine 110, improving our understanding of the food safety of rhodamine dyes.

Herb-Drug Interaction between the Traditional Hepatoprotective Formulation and Sorafenib on Hepatotoxicity, Histopathology and Pharmacokinetics in Rats.

Sorafenib has been used as a standard therapy for advanced hepatocellular carcinoma (HCC). In Asia, patients with HCC are potentially treated with the combination of sorafenib and Chinese herbal medicines to improve the efficiency and reduce the side effects of sorafenib. However, limited information about the herb-drug interactions is available. We hypothesize that the Chinese herbal medicine may exert hepatoprotective effects on the sorafenib-treated group. The aim of this study is to investigate the pharmacokinetic mechanism of drug-drug interactions of sorafenib including interacting with hepatoprotective formulation, Long-Dan-Xie-Gan-Tang formulation (LDXGT) and with two cytochrome P450 3A4 (CYP3A4) inhibitors, grapefruit juice and ketoconazole. Liver enzyme levels and histopathology of liver slices were used to evaluate sorafenib-induced hepatotoxicity and the potential hepatoprotective effects of the LDXGT formulation on subjects treated with the combination of sorafenib and the herbal medicine. In this study, a validated HPLC-photodiode array analytical system was developed for the pharmacokinetic study of sorafenib in rats. As the result of the pharmacokinetic data, pretreatment with the LDXGT formulation did not significantly interact with sorafenib compared with sorafenib oral administration alone. Furthermore, grapefruit juice and ketoconazole did not significantly affect sorafenib metabolism. Furthermore, pretreatment with variable, single or repeat doses of the LDXGT formulation did not suppress or exacerbate the sorafenib-induced hepatotoxicity and histopathological alterations. According to these results, the LDXGT formulation is safe, but has no beneficial effects on sorafenib-induced hepatotoxicity. A detailed clinical trial should be performed to further evaluate the efficacy or adverse effects of the LDXGT formulation in combination with sorafenib in humans.