Carcinoma Xenografts In Nude Mice Research Articles

Effect of overexpression of Rac1 on radiosensitivity of nasopharyngeal carcinoma xenografts in nude mice, and the underlying mechanism

Purpose: To study the effect of overexpression of Rac1 on the radiosensitivity of transplanted nasopharyngeal carcinoma (NPC) in nude mice, and the mechanism involved. 
 Methods: Forty Lucky SPF-grade male thymus-free nude mice were used. Mice were divided into 4 groups: overexpression control, overexpression Rac1, Rac1 inhibition, and inhibition control groups, each with 10 mice. The prepared cell lines were treated with 6 MV x-ray. Before and after radiation, the growth of tumors in each group was monitored. Histomorphological images of nude mice tumors were obtained using hematoxylin and eosin (H&E) stains. Protein expressions of p67, P47, and Rac1 were evaluated bu Western blotting. 
 Results: Transplanted tumor growth slowed down after 20 days. Growth rate was significantly higher in Rac1 and Rac1 overexpression groups than in overexpression and inhibition control groups (p < 0.05). Overexpression of Rac1 resulted in more cell necrosis, incomplete cellular structure, severe nuclear fragmentation, nuclear pyknosis, and cytotoxic red staining in endoscopic tumor tissues (p < 0.05). There were significantly lower expression levels of p67, P47, and Rac1 in Rac1 invasion group than in invasion control and overexpression control groups, while the expression levels of p67 and P47 were significantly higher in overexpression Rac1 group and IR overexpression Rac1 group than in inhibition control (p < 0.05). However, concentrations of p67 and P47 were significantly higher in overexpression Rac1 and IR overexpression Rac1 groups than in inhibition control mice. 
 Conclusion: Rac1 increases the radiosensitivity of NPC xenografts in nude mice via a mechanism related to the regulation of expressions of proteins associated with Rac1/NADPH signaling pathway. Thus, Rac1 is potentially a new target for radio-sensitization of NPC.

Retraction: Isolation of curcumol from zedoary turmeric oil and its inhibitory effect on growth of human hepatocellular carcinoma xenografts in nude mice

Retraction: Isolation of curcumol from zedoary turmeric oil and its inhibitory effect on growth of human hepatocellular carcinoma xenografts in nude mice

RETRACTED ARTICLE]Isolation of curcumol from zedoary turmeric oil and its inhibitory effect on growth of human hepatocellular carcinoma xenografts in nude mice

This study investigated the inhibitory effect and mechanism of curcumol on growth of human hepatocellular carcinoma (HCC) HepG2 xenografts in nude mice. Curcumol was isolated from from zedoary turmeric oil. The HepG2 xenograft model in nude mice was established. Forty modeled nude mice were divided into model, cisplatin, curcumol and cisplatin+curcumol groups. The latter three groups were treated with 2 mg/kg cisplatin, 100 mg/kg curcumol and 2 mg/kg cisplatin combined with 100 mg/kg curcumol, respectively, once every two days, for total seven times. After treatment, compared with cisplatin group, in cisplatin+curcumol group the tumor weight and tumor inhibition rate were obviously increased, the apoptosis rate of HepG2 cells was obviously increased, the expression levels of Caspase-3 and B-cell lymphoma-2 associated X proteins in xenograft tumor were significantly increased, and the expression level of B-cell lymphoma-2 protein was significantly decreased. In addition, the thymus index and spleen index of animal had no significant difference between cisplatin group and cisplatin+curcumol group. In conclusion, the additional use of curcumol can obviously increase the inhibitory effect of cisplatin on growth of HCC HepG2 xenografts in nude mice. The mechanism may be related to the enhanced promotion of apoptosis of tumor.

Antitumor effects of AZD2014, a dual mTORC1/2 inhibitor, against human hepatocellular carcinoma xenograft in nude mice

To investigate the antitumor effects of AZD2014 (a dual mTORC1/2 inhibitor) against human hepatocellular carcinoma (HCC) xenograft in mice. HCCLM3 cells were injected subcutaneously in the right flank of nude mice, and when the tumors were macroscopic, the mice were randomized into 2 groups for daily intraperitoneal injection of AZD2014 (5 mg/kg, n=5) or vehicle (5 mL/kg, n=5) for 24 days. Tumor growth was assessed using calipers every 4 days and the tumor growth curve was drawn. After the final injection, the mice were euthanized and the tumors were dissected for measuring tumor weight and histopathological analysis with HE staining. Immunohistochemical staining was used to detect the expressions of Ki-67, cleaved caspase-3, CD31, and the epithelial-mesenchymal transition (EMT)-related proteins (Ecadherin, N-cadherin, and vimentin) in the tumor tissue. Daily treatment with AZD2014 significantly suppressed HCC growth as compared with the control group. HE staining showed significantly increased tumor necrosis in AZD2014-treated mice. AZD2014 treatment inhibited tumor cell proliferation, angiogenesis and EMT progression as shown by decreased expressions of Ki-67, CD31, N-cadherin, and vimentin and increased expression of E-cadherin in the tumor tissue, and significantly promoted tumor cell apoptosis as shown by an increased expression of cleaved caspase-3 in AZD2014-treated mice. AZD2014 is a highly potent antitumor agent for HCC in nude mice bearing HCC xenografts. AZD2014 can effectively inhibit tumor proliferation, angiogenesis and EMT progression and induce tumor cell necrosis and apoptosis.

Abstract 5510: Mass-action law based general pharmacodynamics theory and algorithm for quantitative computer simulation of drug-evaluations in cells, in animals and in clinical trials

Abstract The Median-Effect Equation (MEE) for single drug, and the Combination Index Equation (CIE) for multiple drug combinations, are basis for general Pharmacodynamics (PD), and Biodynamics (BD). Both, MEE and CIE are derived from system analysis of the Mass-Action Law (MAL). The MAL-PD/BD theory, equations and algorithms allow automated computer simulations for diagnostic plots and quantitative, digital/indexed conclusions. To date, the MAL dynamic theory is cited over 15,000 times, in over 1,248 biomedical journals. The publications include studies in vitro, in cell, in animal and in clinical trial levels, as indicated in Google Scholar Citations -Ting-Chao Chou, or in Pharmacol. Rev. 58: 621-681, 2006 (cited 3,119 times in 914 journals). MEE indicates dose and effect are interchangeable, and all PD Dose-Effect curves (DEC) can be linearized by the Median-Effect Plot (MEP), x= Log (dose) vs y= Log {(fraction affected (fa)/(fraction unaffected (fu)}, where fa+fu=1, and when fa=fu, gives the Median-Effect Dose (Dm). The slope gives “m” value (dynamic order, signifying the shape of DEC), and the Antilog of x-intercept gives the “Dm” value, (signifying the potency of half-affected, such as IC50, ED50, or LD50). The MEE, is the unified theory of the Michaelis-Menten Eq. for enzyme half-saturation, Handerson-Hasselbalch Eq. for pH half-ionization, Hill Eq. for ligand half-occupancy, and the Scatchard Eq, for receptor half-bond and half-free. Since MEP linearizes the DEC, it requires only a minimum of two data pints to simulate entire DEC. Thus, the Two-Data-Point Theory is extremely cost-effective for animal studies and clinical trial protocol designs, as only small number data points is required to achieve efficient computer simulations, with digital/index conclusions. In CIE, CI<1, =1, and >1, quantitatively defines Synergism, Additive Effect, and Antagonism, respectively, for 2 to n drug combs, using CompuSyn. PD requires multiple doses (3-4 doses in animals; 2-3 doses in clinical trials) since single dose yields a data Point, but “a point” which has “no shape”, thus, it is Not a PD in Vivo. For 2-drug-combos using the CI method in vivo for drug A, B, and A+B, require only 10 data points, (3+3+3 plus one control). Earlier examples with CI method in vivo are: 1. Anti-HIV Combos (AZT+IFN, using only 36 AIDS patients), and 2. Anti-cancer Combos (Taxotere+T607, against HCT-116 colon carcinoma xenograft in nude mice), both used only 10 data points and both achieved quantitative determination of Synergism. This report reaffirm Example 2 with the same drug combos against MX-1 mammary carcinoma xenograft in nude mice, using only 10 data points, showed similar synergy at ED75 to ED97, CI= 0.82-0.79 for MX-1, and CI= 0.98-0.64 for HCT-116. Citation Format: Ting-Chao Chou. Mass-action law based general pharmacodynamics theory and algorithm for quantitative computer simulation of drug-evaluations in cells, in animals and in clinical trials [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5510.

Abstract CT155: General pharmacodynamics algorithm-based clinical protocol design with two to three dose-data points for single-drug, and ten data points for two-drug-combination synergy quantification, using computer simulation for digitalized data analysis and conclusions

Abstract The mass-action law (MAL) based Median-Effect Equation (MEE) for single- drug, and the Combination Index Equation (CIE) for drug-combinations, are the basis for unified Pharmacodynamics (PD) and Biodynamics (BD). To date, the MAL-PD/BD theory, in 3 articles alone, have 12,577 citations in 1,248 biomedical journals (Google Scholar Citation-Ting-Chao Chou). The applications mainly in vitro, relatively few in vivo [see, ChouTC, Pharmacol Rev. 58: 621-681, 2006 (cited 3,301 times in 914 journals) or Synergy 1: 3-21, 2014]. In recent presentations at JHU (4.9.2019) and at FDA (5.7.2019 & 11.7.2019), Chou presented, the PD-based clinical protocol design using 2-3 dose-data points for single-drug, and 10 data points for 2-drug combinations synergy quantification, e.g., A (3), B (3), and A+B (3), at constant combination ratio, plus a control. For the MAL-PD/BD based protocol design in vivo (dose-range and dose density, combination ratio), the automated CompuSyn simulation/quantitative PD-parameters (for potency and PD-shape)/diagnostic plots/digitalized conclusions/and automated print-out report, usually takes a few seconds, and print-out may take 1-2 minutes. All drugs (chemical, biological, radiation) should have a Dose-Effect curves (DEC). The MEE indicates that all PD-DEC can be linearized by the Median-Effect Plot (MEP), x= Log (dose) vs y= Log {(fraction affected (fa)/(fraction unaffected (fu)}, where fa+fu=1, and when fa=fu, gives the Median-Effect Dose (Dm). The slope gives “m” value (signifying the shape of DEC), and the antilog of x-intercept gives the “Dm” value, (signifying the potency of half-affected). Since MEP linearizes the DEC, it requires a minimum of two data pints to simulate entire DEC. Thus, the Two-Data-Point Theory is extremely cost-effective for in vivo clinical trial protocol designs, as only small number data points is required to achieve efficient computer simulations, with digital/index conclusions. In CIE, CI<1, =1, and >1, quantitatively defines Synergism, Additive Effect, and Antagonism, respectively, for 2 to n drug combs, using CompuSyn. PD requires multiple doses since “single dose” yields a data-Point, but “a point” has “no shape”, thus, it is never a PD study. Earlier examples with PD-CI method in vivo are: 1. Anti-HIV Combos (AZT+IFN, using only 36 AIDS patients), and 2. Anti-cancer Combos (Taxotere+T607, against HCT-116 colon carcinoma xenograft in nude mice), both used only 10 data points and both achieved quantitative determination of Synergism. This report reaffirm Example 2 with the same drug combos against MX-1 mammary carcinoma xenograft in nude mice, using only 10 data points, which showed similar synergy at ED75 to ED97, CI= 0.82-0.79 for MX-1, and CI= 0.98-0.64 for HCT-116. In conclusion, the MAL-PD/BD and its equations, algorithms and computer simulation provide a rigorous, cost-effective, automated, quantitative biomedical R and D and general drug evaluation guidance in clinical trial design and data analysis, that save resources, time and efforts. Citation Format: Ting-Chao Chou. General pharmacodynamics algorithm-based clinical protocol design with two to three dose-data points for single-drug, and ten data points for two-drug-combination synergy quantification, using computer simulation for digitalized data analysis and conclusions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT155.

Effect of colony‐stimulating factor‐1 receptor overexpression on the growth of nasopharyngeal carcinoma xenografts in nude mice and its mechanism of action

AbstractObjectivesAlthough nasopharyngeal carcinoma (NPC) is sensitive to radiotherapy, local recurrence and distant metastasis still occur in a proportion of patients due to radioresistance. Our previous research confirmed that colony‐stimulating factor‐1 receptor (CSF‐1R) promotes the proliferation, migration, and invasion of 6‐10B cells through the phosphoinositide 3‐kinase (PI3K) pathway in vitro. The objective of the present study was to investigate the effects of colony‐stimulating factor‐1 receptor (CSF‐1R) on proliferation, apoptosis, and autophagy in the human nasopharyngeal carcinoma 6‐10B cell line in vivo, and the possible underlying mechanisms.MethodsA virus carrying the CSF‐1R gene was transfected into 6‐10B cells by lentiviral transfection. A nasopharyngeal carcinoma xenograft model in nude mice was established. Ultimately, the protein expression of proliferation‐, apoptosis‐, autophagy‐, and signaling‐related proteins, such as cyclin D1, B‐cell lymphoma 2 (Bcl‐2), Bcl‐2‐associated X protein, beclin 1, microtubule‐associated protein 1 light chain 3 alpha, sequestosome 1, mechanistic target of rapamycin kinase, PI3K, and protein kinase B (Akt/PKB), in tumor tissues were detected by western blot analysis.ResultsHigh levels of CSF‐1R were expressed in the stably transfected 6‐10B cells, while CSF‐1R promoted the growth of 6‐10B cells in vivo. Cyclin D1, Bcl‐2, microtubule‐associated protein 1 light chain 3 alpha, and beclin 1 were upregulated, while Bcl‐2‐associated X protein and sequestosome 1 were downregulated. Furthermore, the expression of PI3K, phospho‐Akt, Akt, and mechanistic target of rapamycin kinase were upregulated.ConclusionsCSF‐1R overexpression promotes proliferation, reduces apoptosis, and induces autophagy in 6‐10B cells in vivo, and the corresponding mechanism might be executed through activation of the PI3K/Akt pathway.

Effects of vitamin K2 on the growth and expression of HDGF in transplanted tumor of human liver cancer in nude mice

Objective To investigate the effects of vitamin K2 on the growth of transplanted tumor and the expression of HDGF in human hepatocellular carcinoma in nude mice. Methods After the establishment of metastatic human hepatocellular carcinoma models, give the tumor-bearing nude mice oral vitamin K2, the nude mice were randomly divided into three groups, 8 nude mice in each group: control group, vitamin K2 low concentration group [20 mg/(kg·d)], and vitamin K2 high concentration group [45 mg/(kg·d)]. Tumor size, tumor weight were measured during administration, inhibitory rate was calculated. The expression of HDGF protein in transplanted tumor tissues was detected after different concentrations of vitamin K2. Results The inhibitory rate of control group, low vitamin K2 concentration group and high vitamin K2 concentration group was 0%, (36.13±4.38)%, (57.90±2.57)%, respectively. Compared with the control group, the vitamin K2 intervention group showed significantly reduced tumor tissue and increased inhibitory rate (t=25.460, 80.610, P<0.05). Western blotting analysis showed that the grayscale analysis values of HDGF protein expression in low vitamin K2 concentration group and high vitamin K2 concentration group were (0.634±0.030) and (0.278±0.040), which significantly inhibited the expression of HDGF protein in tumor cells of tumor-bearing nude mice, and were statistically significant compared with the control group (t=32.730, 49.520, P<0.05). Conclusion Vitamin K2 inhibited the growth of human hepatocellular carcinoma xenografts in nude mice and down-regulated the expression of HDGF in tumor cells. Key words: Vitamin K2; Nude mice beared human hepatocellular carcinoma; Hepatoma-derived growth factor

Computerized quantification of drugs synergism in animal studies or in clinical trials using only ten data points

Abstract The median-effect equation (MEE) derived from the mass-action law (MAL) is the unified theory of dose-effect pharmacodynamics (PD) and biodynamics (BD). MEE enables the linearization of a dose-effect curve into a straight linedefined by two data points. Thus any two data points can represent an entire dose-effect curve. For dose-effect curves using MAL, dose-zero can serve as 3rd point, and the universal reference point, the median-effect dose (Dm) serves as 4th point. This functionality has tremendous significance for in vivo studies. Fewer data points are required for a dose-effect curve, facilitating economical and ethically sustainable PD analyses. The extension of MEE from a single drug to multiple drugs establishes the general combination index equation (CIE), which quantitatively defines synergism (CI 1]. Although the CI method is often (>6000 citations) applied in in vitro studies, it is rarely used in animal studies or clinical trials. In vivo drug combination studies that use only single dose or statistical p value analyses do not allow quantitative synergy claims. This article presents two examples for drug combinations in vivo: (i) in animals (anticancer drug combination against human HCT-116 colon carcinoma xenografts in nude mice, Taxotere + T607) and (ii) in a clinical trial (anti-retroviral drug combinations against HIV/AIDS, AZT + INF). Only 36 patients respectively only 66 nude mice were required. Both examples require only ten data points (D1, D2 and [D1+D2], each with 3 doses plus one control) to quantitatively determine synergism or antagonism with the CompuSyn software.

Preclinical evaluation of novel PI3K/mTOR dual inhibitor SN202 as potential anti-renal cancer agent

ABSTRACTThe PI3K/mTOR pathway is one of the most frequently aberrantly activated pathways in human malignancies, such as renal cell carcinoma (RCC), and contributes to resistance to antitumor therapies. Thus, PI3K/mTOR is an attractive target for the development of antitumor agents. In this study, we evaluated the preclinical effects of a novel inhibitor SN202. We examined Akt/mTOR activities in renal cancer cells after SN202 treatment. The preclinical effects of SN202 on tumor growth were evaluated in renal cancer cells in vitro and in murine xenografts in vivo. SN202 inhibits PI3Kα, PI3Kγ, and mTOR, the corresponding IC50 values were 3.2, 3.3, and 1.2 nM, respectively. In A498, 786-0, and ACHN renal cancer cell lines, SN202 inhibits cell proliferation in a dose-dependent manner and significantly inhibits 786-0 cell growth. Western blot analysis revealed that SN202 decreases the phosphorylation of PI3K downstream signaling molecules, Akt and S6K, in 786-0 renal cancer cells. Furthermore, oral administration of SN202 results in significant inhibition in human renal carcinoma xenografts in nude mice and favourable pharmacokinetic properties in rats. These results suggest that SN202 might be a promising therapeutic agent against RCC as a dual PI3K/mTOR inhibitor.

Abstract 3945: CBT-102, an oral multi-kinase small molecule, demonstrates favorable activity in human hepatocellular carcinoma animal models and cardiac safety in rabbit Purkinje and beagle dogs compared to sorafenib

Abstract Background: Cancer is a complex disease involving disruption of more than one receptor or signaling pathway. Inhibiting multiple oncogenic targets have been shown to be an important strategy in managing relapse and resistance. CBT-102 is an oral mTKI inhibiting several key oncogenic drivers including angiogenesis via VEGFR and PDGFR, MAPK pathway via B-RAF and C-RAF, in addition to inhibiting RET, CSF1R, c-KIT, and FLT3. Antitumor activity of CBT-102 was compared to sorafenib in two human primary HCC carcinoma xenografts in nude mice (LIMsh050 and PLCPRF5) and cardiac safety was evaluated in rabbit heart Purkinje fibers and in conscious telemeterized beagle dogs. Methods: Female BALB/c nude mice were inoculated with LIMsh050 and PLCPRF5 tumors to establish the tumor models. In the LIMsh050 model, CBT-102 was administered orally QD x 21 days at 4, 10, and 25 mg/kg and sorafenib at 10 mg/kg (n=8/group). In the PLCPRF5 model, CBT-102 was administered orally QD x 14 days at 3, 8, and 20 mg/kg vs. 8 and 20 mg/kg sorafenib group (n=7/group). In both experiments, in addition to tumor volume, body weight and mortality were monitored. For the cardiac safety studies, rabbit Purkinje fibers were isolated from adult rabbit right heart perfused for 10 minutes with vehicle control, CBT-102 groups (1, 3, and 10 µM) or sorafenib and changes in action potentials were measured. In the second study, ECG parameters were measured after single oral administration of CBT-102 at 15, 45, and 67.5 mg/kg to beagle dogs (n=6/group) via telemetry for 24 h. Results: In both the LIMsh050 and PLCPRF5 models, CBT-102 and sorafenib doses were well tolerated with no suspension of dosing due to weight loss or deaths. CBT-102 demonstrated inhibition of tumor growth in both models, to a significantly greater extent than sorafenib at comparable doses. In the PLCPRF5 model, CBT-102 demonstrated an ED50 of 2.5 mg/kg vs. 16.8 mg/kg of sorafenib. Importantly, unlike sorafenib, CBT-102 did not show a cardiac liability: there were no changes in action potential duration in rabbit Purkinje fibers nor any changes in cardiac conduction parameters (HR, QTc, RR, PR, and QRS intervals) in beagle dogs. Conclusions: CBT-102 demonstrated improved efficacy in HCC xenografts models compared to sorafenib and does not appear to have the cardiac liability observed with sorafenib. CBT-102 will advance into GLP toxicology studies with a potential IND filing in 2H 2018. Citation Format: Sarath Kanekal, Xiquan Zhang, Yanrui Song, Tan Wenmiao, Juan Zhang, Deyi Zhang, Henry Li, Mamatha Reddy, Gavin Choy, Sanjeev Redkar. CBT-102, an oral multi-kinase small molecule, demonstrates favorable activity in human hepatocellular carcinoma animal models and cardiac safety in rabbit Purkinje and beagle dogs compared to sorafenib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3945.

Synthesis and Evaluation of the Tumor Cell Growth Inhibitory Potential of New Putative HSP90 Inhibitors.

Background: Heat shock protein 90 (HSP90) is a well-known target for cancer therapy. In a previous work, some of us have reported a series of 3-aryl-naphtho[2,3-d]isoxazole-4,9-diones as inhibitors of HSP90. Methods: In the present work, various compounds with new chromenopyridinone and thiochromenopyridinone scaffolds were synthesized as potential HSP90 inhibitors. Their binding affinity to HSP90 was studied in vitro. Selected compounds (5 and 8) were further studied in various tumor cell lines regarding their potential to cause cell growth inhibition, alter the cell cycle profile, inhibit proliferation, and induce apoptosis. Their effect on HSP90 client protein levels was also confirmed in two cell lines. Finally, the antitumor activity of compound 8 was studied in A431 squamous cell carcinoma xenografts in nude mice. Results: Our results indicated that treatment with compounds 5 and 8 decreased the proliferation of tumor cell lines and compound 8 induced apoptosis. In addition, these two compounds were able to downregulate selected proteins known as “clients” of HSP90. Finally, treatment of xenografted mice with compound 5 resulted in a considerable dose-dependent inhibition of tumor growth. Conclusions: Our results show that two new compounds with a chromenopyridinone and thiochromenopyridinone scaffold are promising putative HSP90 inhibitors causing tumor cell growth inhibition.

Apigenin sensitizes hepatocellular carcinoma cells to doxorubic through regulating miR-520b/ATG7 axis

Chemo-resistance is a serious obstacle for successful treatment of cancer. Apigenin, a dietary flavonoid, has been reported as an anticancer drug in various malignant cancers. This study aimed to investigate the potential chemo-sensitization effect of apigenin in doxorubicin-resistant hepatocellular carcinoma cell line BEL-7402/ADM. We observed that apigenin significantly enhanced doxorubicin sensitivity, induced miR-520b expression and inhibited ATG7-dependent autophagy in BEL-7402/ADM cells. In addition, we also showed that miR-520b mimics increased doxorubicin sensitivity and inhibited ATG7-dependent autophagy. Meanwhile, we indicated that ATG7 was a potential target of miR-520b. Furthermore, APG inhibited the growth of hepatocellar carcinoma xenografts in nude mice by up-regulating miR-520b and inhibiting ATG7. Our finding provides evidence that apigenin sensitizes BEL-7402/ADM cells to doxorubicin through miR-520b/ATG7 pathway, which furtherly supports apigenin as a potential chemo-sensitizer for hepatocellular carcinoma.

Berberine binds RXR\u03b1 to suppress \u03b2-catenin signaling in colon cancer cells

Berberine, an isoquinoline alkaloid, is a traditional oriental medicine used to treat diarrhea and gastroenteritis. Recently, we reported that it could inhibit the growth of intestinal polyp in animals and in patients with the familial adenomatous polyposis by downregulating β-catenin signaling. However, the intracellular target mediating the effects of berberine remains elusive. Here, we provide evidence that berberine inhibits β-catenin function via directly binding to a unique region comprising residues Gln275, Arg316 and Arg371 in nuclear receptor retinoid X receptor alpha (RXRα), where berberine concomitantly binding to and synergistically activating RXRα with 9-cis-retinoic acid (9-cis-RA), a natural ligand binding to the classical ligand-binding pocket of RXRα. Berberine binding promotes RXRα interaction with nuclear β-catenin, leading to c-Cbl mediated degradation of β-catenin, and consequently inhibits the proliferation of colon cancer cells. Furthermore, berberine suppresses the growth of human colon carcinoma xenograft in nude mice in an RXRα-dependent manner. Together, our study not only identifies RXRα as a direct protein target for berberine but also dissects their binding mode and validates that berberine indeed suppresses β-catenin signaling and cell growth in colon cancer via binding RXRα, which provide new strategies for the design of new RXRα-based antitumor agents and drug combinations.

Abstract 4554A: Simple, efficient, and quantitative approach for determination of synergism, additive effect, and antagonism of drugs in vivo using combination index method: a proposition for clinical protocol design and regulatory synergy claims

Abstract Based on the systematic dynamic rate derivations with the mass-action principle and mathematical induction-deduction, the median-effect equation of Chou, and the combination index (CI) equation and their algorithm allow quantitative determination of synergism (CI&amp;lt;1), additive effect (CI=1) and antagonism (CI&amp;gt;1) (Pharmacol Rev 58:621-681, 2006). Based on Google Scholar Citations, the CI simulation has been cited 7,712 times in &amp;gt;800 bio-medical journals as of 10.19.2016. However, most citing papers were with experiments in vitro, relative few in vivo and rarely in clinical trials. This is despite the fact that the CI method equation, algorithm, and the CompuSyn software are the same for in vitro and in vivo, although the practical aspects such as sample size, time and cost, the readiness of the end-point measurement, and the ethical considerations are different. Our recent publication (in Fu J et al. Synergy 3: 15-30, 2016) of two-drug combination (docetaxel and T-607 compound, 1:2) against HCT-116 colon carcinoma xenograft in nude mice has demonstrated very desirable synergy. We now illustrate the experimental and data-analytical details of the experimental planning and design, execution, measurements, for docetaxel and T-Compound combination against MX-1 mammary carcinoma xenograft in nude mice. Treatment Q3Dx4 i.v. starting Day 7, and the tumor sizes and bodyweight changes measured on Days 16, 19, and 22. A total of 50 nude mice were used (each drug and combinations, 3 doses each (n=5) plus a control group of 5). Full spectrum of synergism (CI=0.79 to 0.93) were observed on Days 19 and 22. Automated simulation were carried out for dose-effect curve, ME plot, Fa-CI plot, isobologram, and Fa-DRI (Dose-Reduction Index) plot with CompuSyn software (Free download available upon registration via www.combosyn.com). This simple, efficient, “econo-green” bio-research paves the way for drug combination clinical trial design and regulatory synergy claims. Citation Format: Jianing Fu, Ting-Chao Chou. Simple, efficient, and quantitative approach for determination of synergism, additive effect, and antagonism of drugs in vivo using combination index method: a proposition for clinical protocol design and regulatory synergy claims [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4554A. doi:10.1158/1538-7445.AM2017-4554A

Abstract 4554: Unified theoretical algorithms for graphics dynamic multiple transformations of dose-effect relationships with computer simulation

Abstract Based on physico-chemical principle of the mass-action law, bio-system analysis (arrangement &amp; combinatorial) several hundred rate equations have been derived and published. Mathematical induction allows the deduction of them into the unified general median-effect equation (MEE) that makes dose &amp; effect “interchangeable” for the 1st order and higher order dynamics. Based on the MEE plot, all dose-effect curves can be “linealized” which allows the quantitative determination of x-intercept (log Dm which signifies potency) and slope (m which signifies shape or dynamic order). Extension of MEE to multi-drug combinations resulted the combination index (CI) theorem which quantifies synergism (CI&amp;lt;1), additive effect (CI=1), and antagonism (CI&amp;gt;1). This unprecedented approach and multi-step logics have been summarized in [Chou TC. Pharmacol Rev 58: 621-681, 2006] which has been cited 1,261 times in 485 bio-medical journals (http://www.researcherid.com/rid/B4111-2009 as of 10.19.2016). Dose-effect curves (single &amp; multi-drugs) can be quantitatively transformed into the MEE plot, Fa-CI plot, isobologram, Fa-DRI Plot for dose-reduction index, and when n&amp;gt;2, the polygonogram. This presentation will illustrate of combinations (1:5) of docetaxel (TXT) with T-compound against HCT-116 colon carcinoma xenograft in nude mice. In all, only 66 nude mice have been used, i.e. TXT 3 doses, T 3 doses and TXT+T 4 doses, plus a control group, each N=6. The MEE “Linearization” allows the “Econo-Green Bio-research”, with small experimental size and small number of data points, using small number of animals. The algorithms based computer simulation using CompuSyn software can be completed within one second after the dose and effect data entries. The CompuSyn has been offered for free download upon registration via www.combosyn.com. Note: This abstract was not presented at the meeting. Citation Format: Ting-Chao Chou. Unified theoretical algorithms for graphics dynamic multiple transformations of dose-effect relationships with computer simulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4554. doi:10.1158/1538-7445.AM2017-4554

The role (if any) of chemotherapy in hepatocellular carcinoma

The study by Masatoshi Kudo and colleagues 1 Kudo M Moriguchi M Numata K et al. S-1 versus placebo in patients with sorafenib-refractory advanced hepatocellular carcinoma (S-CUBE): a randomised, double-blind, multicentre, phase 3 trial. Lancet Gastroenterol Hepatol. 2017; (published online April 6.)http://dx.doi.org/10.1016/S2468-1253(17)30072-9 Google Scholar published in The Lancet Gastroenterology & Hepatology is an appreciated effort to overcome the unmet needs in treatment of advanced hepatocellular carcinoma. The study is a well-designed, randomised, double-blind, multicentre phase 3 trial comparing S-1 with placebo after progression on or intolerance to sorafenib in Japanese patients with advanced hepatocellular carcinoma. The results unfortunately did not show an improvement in survival with S-1 (11·1 months for S-1 and 11·2 months for placebo) despite encouraging phase 1/2 data (progression-free survival of 3·7 months and overall survival of 16·6 months). 2 Furuse J Okusaka T Kaneko S et al. Phase I/II study of the pharmacokinetics, safety and efficacy of S-1 in patients with advanced hepatocellular carcinoma. Cancer Sci. 2010; 101: 2606-2611 Crossref PubMed Scopus (35) Google Scholar A post-hoc exploratory predictive analysis detected an enriched population with significant benefit (overall survival of 14·0 months for S-1 versus 12·3 months for placebo, p=0·016). These patients had Child-Pugh A cirrhosis, alpha-fetoprotein less than 400 ng/mL, and stage III or IV disease. However, there was no mention of correlation between outcome and prior sorafenib exposure, or if S-1 activity is potentiated by sorafenib-induced tumour necrosis, 3 Itashiki Y Harada K Ferdous T Yoshida H Effects of tumor necrosis factor-related apoptosis-inducing ligand alone and in combination with fluoropyrimidine anticancer agent, S-1, on tumor growth of human oral squamous cell carcinoma xenografts in nude mice. Anticancer Res. 2007; 27: 2365-2375 PubMed Google Scholar although adding S-1 to sorafenib in the first-line setting did not show added benefit compared with historical results with sorafenib alone (overall survival 10·5 months). 4 Ooka Y Chiba T Ogasawara S et al. A phase I/II study of S-1 with sorafenib in patients with advanced hepatocellular carcinoma. Invest New Drugs. 2014; 32: 723-728 Crossref PubMed Scopus (10) Google Scholar S-1 versus placebo in patients with sorafenib-refractory advanced hepatocellular carcinoma (S-CUBE): a randomised, double-blind, multicentre, phase 3 trialS-1 did not prolong overall survival in patients with sorafenib-refractory advanced hepatocellular carcinoma. Further research is needed to identify subgroups of patients who might benefit from S-1. Full-Text PDF

Effect of Six1,TGF-β,VEGF-C that promoting tumor lymphangiogenesis in human laryngeal carcinoma xenografts in nude mice

Objective:To research the effect of Six1,TGF-β,VEGF-C that promoting tumor lymphangiogenesis in human laryngeal carcinoma xenografts in nude mice. Method:Technology of RNA interference was used for silencing Six1 and TGF-β genes expression in laryngeal squamous cell, preparation Six1-targeting and TGF-β-targeting siRNA for transfecting into laryngeal squamous cell carcinoma, sieve out positive clone cell and amplify. Preparing bearing cancer mice, the mice were divided into five groups, group A (untransfected),group B(empty vector), group C(Six1-siRNA),group D(TGF-β-siRNA),group E(Six1+TGF-β-siRNA). 6-12mm when the tumor has grown, the mice were sacrificed by cervical. The size of each tumor and metastasis were observed and recorded.The protein expression of Six1,TGF-β and VEGF-C was determined by immunohistochemistry and Western blot.The mRNA of Six1,TGF-β and VEGF-C was determined by RT-PCR. Result:The average tumor volume and the number of metastasis cases in group B have no statistically significant compared with group A. The average tumor volume in group C, group D and group E has no significantly reduced, but there is a clear reduction of the number of metastasis cases compared with group A. The average tumor volume and number of metastasis cases in group E has no significantly reduced compared with group C and group D. Both protein and mRNA expression of Six1, TGF-β and VEGF-C in group B had no significant difference compared with group A. In group C, group D and group E,both protein and mRNA expression of VEGF-C was decreased,difference has statistically significant compared with group A. Both protein and mRNA expression of VEGF-C in group E had no significant difference compared with group C and group D. Conclusion:Both Six1 and TGF-β can mediate tumor lymphangiogenesis and lymph node metastasis by mediate the expression of VEGF-C. Suggest that Six1,TGF-β might be a potential therapeutic target for preventing lymph node metastasis of tumor.

Metformin inhibits the growth of hypopharyngeal carcinoma xenografts in nude mice model

Objective:To investigated the effect of metformin on growth inhibition of hypopharyngeal carcinoma xenograft in nude mice and to study the theoretical basis of metformin for the treatment of hypopharyngeal carcinoma in clinical.Method:①To observe the course of metformin treatment, there were no severe adverse effects among the xenograft mice. ②After 28 days treatment, the average volumes of hypopharyngeal carcinoma xenografts of the metformin treated groups were significantly low compared with that of the control group.〖JP2〗The inhibition ratio of metformin at the concentration of 40 mg/(kg•d) and 200 mg/(kg•d) to the growth of tumor was 23.30%±7.17% and 40.51%±9.25%, respectively.③The aver ageoptical density(OAD) of AMPK in tumors of the control, met40 and met200 group was 0.246±0.038, 0.262±0.046, and 0.308±0.087, respectively. The OAD of p21 was 0.288±0.065, 0.305±0.091, and 0.477±0.114, respectively. And the OAD of cyclin D1 was 1.608±0.101, 0.781±0.055, and 0.711±0.046, respectively. Both the OAD of AMPK and p21 in met40 and met200 group were observably higher than those of the control group (P< 0.05). But the OAD of cyclin D1 in met40 and met200 group were remarkably lower than the control group (P< 0.05). ④The Western Blot drawed the consistant trend variation with immunohistochemical analyses. The gray value of AMPK in tumors of the control,met40 and met200 group was 0.641±0.042, 1.124±0.085, and 1.783±0.128; The gray value of p21 was 0.474±0.027, 0.594±0.063, and 0.970±0.079; The gray value of cyclin D1 was 1.608±0.101,0.781±0.055, and 0.711±0.046.Result:①To observe the course of metformin treatment, there were no severe adverse effects among the xenograft mice. ②After 28 days treatment, the average volumes of hypopharyngeal carcinoma xenografts of the metformin treated groups were significantly low compared with that of the control group.〖JP2〗The inhibition ratio of metformin at the concentration of 40 mg/(kg•d) and 200 mg/(kg•d) to the growth of tumor was 23.30%±7.17% and 40.51%±9.25%, respectively.③The aver ageoptical density(OAD) of AMPK in tumors of the control, met40 and met200 group was 0.246±0.038, 0.262±0.046, and 0.308±0.087, respectively. The OAD of p21 was 0.288±0.065, 0.305±0.091, and 0.477±0.114, respectively. And the OAD of cyclin D1 was 1.608±0.101, 0.781±0.055, and 0.711±0.046, respectively. Both the OAD of AMPK and p21 in met40 and met200 group were observably higher than those of the control group (P< 0.05). But the OAD of cyclin D1 in met40 and met200 group were remarkably lower than the control group (P< 0.05). ④The Western Blot drawed the consistant trend variation with immunohistochemical analyses. The gray value of AMPK in tumors of the control,met40 and met200 group was 0.641±0.042, 1.124±0.085, and 1.783±0.128; The gray value of p21 was 0.474±0.027, 0.594±0.063, and 0.970±0.079; The gray value of cyclin D1 was 1.608±0.101,0.781±0.055, and 0.711±0.046.Conclusion:Metformin can suppress the growth of hypopharyngeal carcinoma xenograft in nude mice and inducing tumor cell apoptosis without any severe adverse effects.

Houttuynia cordataThunb Promotes Activation of HIF-1A–FOXO3 and MEF2A Pathways to Induce Apoptosis in Human HepG2 Hepatocellular Carcinoma Cells

Houttuynia cordata Thunb (H cordata), a medicinal plant, has anticancer activity, as it inhibits cell growth and induces cell apoptosis in cancer. However, the potential anti-cancer activity and mechanism of H cordata for human liver cancer cells is not well understood. Recently, we identified hypoxia-inducible factor (HIF)-1A, Forkhead box (FOX)O3, and MEF2A as proapoptotic factors induced by H cordata, suggesting that HIF-1A, FOXO3, and MEF2A contribute to the apoptosis of HepG2 hepatocellular carcinoma cells. FOXO3 transcription factors regulate target genes involved in apoptosis. H cordata significantly increased the mRNA and protein expression of HIF-1A and FOXO3 and stimulated MEF2A expression in addition to increased apoptosis in HepG2 cells within 24 hours. Therefore, we determined the potential role of FOXO3 on apoptosis and on H cordata–induced MEF2A in HepG2 cells. HIF-1A silencing by siRNA attenuated MEF2A and H cordata–mediated FOXO3 upregulation in HepG2 cells. Furthermore, H cordata–mediated MEF2A expression enhanced caspase-3 and caspase-7, which were abolished on silencing FOXO3 with siRNA. In addition, H cordata inhibited growth of human hepatocellular carcinoma xenografts in nude mice. Taken together, our results demonstrate that H cordata enhances HIF-1A/FOXO3 signaling, leading to MEF2A upregulation in HepG2 cells, and in parallel, it disturbs the expression of Bcl-2 family proteins (Bax, Bcl-2, and Bcl-xL), which results in apoptosis. Taken together, these findings demonstrate that H cordata promotes the activation of HIF-1A–FOXO3 and MEF2A pathways to induce apoptosis in human HepG2 hepatocellular carcinoma cells and is, therefore, a promising candidate for antitumor drug development.