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Exploring Anticancer Potential of Camptothecin Isolated from Nothapodytes Nimmonianain the Treatment of Prostate and Lung Carcinoma
Abstract
Background: Cancer is a life-threatening disease which is a major threat to global health which continues to affect predominantly in developing nations. Present research work has been oriented towards the determination of the anticancer potential of camptothecin (CPT) as herbal medicine for the treatment of prostate and lung carcinoma. Methods: Structural determination of camptothecin has been performed by different analytical techniques where anticancer potential was tested by MTT assay, Flow cytometry and DAPI on A549 and LNCaP cells lines. Results: FTIR spectra of camptothecin showed peaks related to specific structure which is nearly equal to standard structure of CPT. NMR spectra of camptothecin showed specific peaks in the region of delta 8.686 - 5.279, the signals of H-7 related to structural features similar to camptothecin. LCMS spectra of camptothecin showed mean retention time at 3.620 and covered 100 % area along with mass spectra gives precursor m/z peak at 349.2 [M+H]+ matches to standard molecular weight of camptothecin. CPT has been used as competent alternative to systemic chemotherapy to cure lung and prostate carcinoma having IC50 value 3.421 μg /ml and 5.253μg /ml respectively. CPT successfully induces apoptosis in A549 and LNCaP cell lines 72.12 ± 3.45 % and 66.41 ± 4.50 % as compared to control 4.28 ± 1.78 and 1.52 ± 0.58 respectively which was proved by DAPI and flow cytometry. Conclusions: Chemical fingerprinting and structural elucidation confirmed that isolated moiety was camptothecin and it has great potential in treatment of lung and prostate carcinoma as a competent alternative to chemotherapy in the form of herbal medicine.
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Background
Objective of present research work is to develop and validate cost-effective analytical tool for determination of camptothecin (CPT) and determine its anticancer potential against prostate cancer LNCaP cell lines . Structural elucidation has been performed by mass spectrometry, Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, and MTT assay utilized for in vitro cytotoxicity where spectrometric method was used for estimation of camptothecin.
Results
Mass spectra showed peak at 349.2 which matches to standard molecular weight of camptothecin. FTIR and NMR spectra conformed functional moieties and structure of isolated camptothecin which was nearly equal to values mentioned in standard structure of camptothecin. IC 50 values of CPT against LNCaP cell lines was found to be 3.561 μg/ml. Lambda max of CPT was found to be at 225 nm and calibration curve found to be linear over the concentration range from 2 to 70 μg/ml of camptothecin. Developed method was found to be linear, accurate, and precise. LOD and LOQ were found to be 0.0524 μg/ml and 0.1614 μg/ml, respectively. Developed method has % relative standard deviation less than one which is reproducible hence % recovery was found to be 99.80%.
Conclusions
FTIR, NMR, and mass spectrometry results conforms isolated compound was camptothecin; cytotoxicity study proves it has strong potential in treatment of prostate carcinoma as competent alternative to chemotherapy in the form of herbal medicine and the developed UV method proves to be valid, sensitive, and applicable for rapid, accurate, precise, and economical determination of camptothecin.
Background
The liver is the vital organ which plays a major role in metabolism with numerous functions in the human beings such as protein synthesis, hormone production, and detoxification. Present research work is focused on hepatoprotective potential of chloroform (PNFC) and ethyl acetate (PNFEA) endophytic fractions from Phyllanthus niruri Linn . against CCl 4 -induced hepatotoxicity in albino Wistar rats. To test our hypothesis, both endophytic fungal fractions were tested for vitro antioxidant and in vivo hepatoprotective activity. Serum biochemical parameters like SGOT, SGOT, SALP, cholesterol, bilirubin, and protein were estimated to assess hepatoprotective activity.
Results
Group of rats treated with CCl 4 possess marked hepatic damage and oxidative stress which indicates that cellular leakage and loss of functional integrity of cell membrane in liver. PNFC and PNFEA fractions of endophyte from Phyllanthus niruri Linn . stem have significantly reduced the elevated levels of biomarkers like SGPT, SGOT, SALP, bilirubin, cholesterol, and total protein in CCl 4 -induced hepatotoxicity in rats. The results obtained confirm hepatoprotective activity of endophytic fractions (PNFC and PNFEA) mediated through the stabilization of plasma membrane, repair of hepatic tissue damage, return of biochemical marker levels to normal, and regeneration of hepatocytes. Histopathological observations revealed improvement in the liver architecture after the treatment of secondary metabolites of endophytic fractions against CCl 4 -induced liver damage. Both fungal endophytes PNFC and PNFEA showed DPPH scavenging activity with IC 50 of 97.79 μg/ml and 108.40 μg/ml, respectively, and possess antioxidant potential. Presence of flavonoids in the both fractions of endophytes may be a possible reason for its antioxidant potential and identified as Eurotium amstelodami strain.
Conclusion
Both fungal endophytes PNFC and PNFEA possess hepatoprotective potential due to the presence of secondary metabolites of fungi, i.e., Eurotiumam stelodami strain which support the claim endophytes and act as a potent biomedicine for treatment of various chronic diseases.
This research work was carried out to measure the gastro retentive efficacy of micro sponges via optimization of targeted floating lafutidine microsponges for improved site specific absorption for gastric ulcer. Modified quasi emulsion solvent diffusion process was used to fabricate microsponges. The effect of different levels of ethyl cellulose and polyvinyl alcohol concentration, selected as independent variables was determined on the % entrapment efficiency, % buoyancy and % cumulative drug release. Modified rosette rise apparatus was used for In vitro release and the release data best fitted zero order models and mechanism of drug release is independent of concentration of drug. The optimized formulation (MS5) demonstrated favourable % entrapment efficiency (92.83 ± 1.8), % buoyancy (86.30 ± 1.8) and % cumulative drug release (85.7 ± 1.2). SEM revealed spherical and porous micro sponges. DSC confirmed molecular dispersion of the drug in the microsponge' polymeric matrix. This study presents a new approach based on floating ability of microsponge for treatment of gastric ulcer. Abstract Introduction
Background
The objective of present research work is to design and characterize camptothecin gel using Carbopol-934 for the treatment of epidermoid carcinoma. Optimized herbal gel formulations were evaluated for homogeneity and appearance, viscosity, extrudability, spreadability, drug content, drug release, pH, and in vitro skin cancer activity on A431 cell lines.
Results
Mass and Infrared Spectra respectively conforms molecular weight and functional groups present in camptothecin. All the formulations F1 to F5 showed good homogeneity, pH from 6.68 to 6.90, spreadability in the range of 15.81–23.37 gm.cm/s, extrudability 85.51–90.45% w/w, drug content 89.12–96.64%, and in vitro diffusion 88.36–98.40%, respectively. The drug release study showed that all the formulations followed a diffusion-controlled, zero-order release mechanism. Anticancer activity results indicate that camptothecin gel induce cell death in A-439 cells having IC 50 48.03 μg and % apoptosis 54.67 ± 4.58.
Conclusion
Topical delivery of camptothecin alleviates the side effects caused by systemic chemotherapy; hence, the developed herbal gel formulation can be effectively useful to deliver camptothecin in the treatment of epidermoid carcinoma on A-431 cells.
Background:
Camptothecin (CPT) and its derivatives are currently used as second- or third-line treatment for patients with endocrine-resistant breast cancer (BC). These drugs convert nuclear enzyme DNA topoisomerase I (TOP1) to a cell poison with the potential to damage DNA by increasing the half-life of TOP1-DNA cleavage complexes (TOP1cc), ultimately resulting in cell death. In small and non-randomized trials for BC, researchers have observed extensive variation in CPT response rates, ranging from 14 to 64%. This variability may be due to the absence of reliable selective parameters for patient stratification. BC cell lines may serve as feasible models for generation of functional criteria that may be used to predict drug sensitivity for patient stratification and, thus, lead to more appropriate applications of CPT in clinical trials. However, no study published to date has included a comparison of multiple relevant parameters and CPT response across cell lines corresponding to specific BC subtypes.
Method:
We evaluated the levels and possible associations of seven parameters including the status of the TOP1 gene (i.e. amplification), TOP1 protein expression level, TOP1 activity and CPT susceptibility, activity of the tyrosyl-DNA phosphodiesterase 1 (TDP1), the cellular CPT response and the cellular growth rate across a representative panel of BC cell lines, which exemplifies three major BC subtypes: Luminal, HER2 and TNBC.
Results:
In all BC cell lines analyzed (without regard to subtype classification), we observed a significant overall correlation between growth rate and CPT response. In cell lines derived from Luminal and HER2 subtypes, we observed a correlation between TOP1 gene copy number, TOP1 activity, and CPT response, although the data were too limited for statistical analyses. In cell lines representing Luminal and TNBC subtypes, we observed a direct correlation between TOP1 protein abundancy and levels of enzymatic activity. In all three subtypes (Luminal, HER2, and TNBC), TOP1 exhibits approximately the same susceptibility to CPT. Of the three subtypes examined, the TNBC-like cell lines exhibited the highest CPT sensitivity and were characterized by the fastest growth rate. This indicates that breast tumors belonging to the TNBC subtype, may benefit from treatment with CPT derivatives.
Conclusion:
TOP1 activity is not a marker for CPT sensitivity in breast cancer.
The aim of present research work to formulate microsponge of Amphotericin B to control the delivery rate of Amphotericin B to a predetermined site in human body with with control and maximum therapeutic drug delivery. Controlled release of drugs onto the epidermis with assurance that the drug remains primarily localized and does not enter the systemic circulation is the biggest challenge. To overcome this we Developed microsponges of amphotericin-B evaluated for compatibility studies, morphology and surface topography, Particle size, Percentage yield, Loading efficiency, Drug release, Diffusion study and antimicrobial activity. The prepared microsponge of amphotericin-B was found to be satisfactory. Formulation F2 of Amphotericin B wasbest in the class of Eudragit RS- 100 as a retarding polymer. The developed microsponges improve therapeutic potential of antifungal drug amphotericin-B in the treatment of Candida albicans and Aspargillus fungates and it is proved by antifungal activity. © 2019 Yerkure Tanitim ve Yayincilik Hizmetleri A.S. All rights reserved.
Historically, the prognosis for individuals diagnosed with lung cancer has been bleak. However, the past 10 years have seen important advances in treatment and diagnosis which have translated into the first improvements seen in lung cancer survival. This review highlights the major advances in treatments with curative intent, systemic targeted therapies, palliative care and early diagnosis in lung cancer. We discuss the pivotal research that underpins these new technologies/strategies and their current position in clinical practice.
Cancer is a frightful disease and represents one of the biggest health-care issues for the human race and demands a proactive strategy for cure. Plants are reservoirs for novel chemical entities and provide a promising line for research on cancer. Hitherto, being effective, chemotherapy is accompanied by certain unbearable side effects. Nevertheless, plants and plant derived products is a revolutionizing field as these are Simple, safer, eco-friendly, low-cost, fast, and less toxic as compared with conventional treatment methods. Phytochemicals are selective in their functions and acts specifically on tumor cells without affecting normal cells. Carcinogenesis is complex phenomena that involves many signaling cascades. Phytochemicals are considered suitable candidates for anticancer drug development due to their pleiotropic actions on target events with multiple manners. The research is in progress for developing potential candidates (those can block or slow down the growth of cancer cells without any side effects) from these phytochemicals. Many phytochemicals and their derived analogs have been identified as potential candidates for anticancer therapy. Effort has been made through this comprehensive review to highlight the recent developments and milestones achieved in cancer therapies using phytomolecules with their mechanism of action on nuclear and cellular factors. Furthermore, drugs for cancer treatment and their limitations have also been discussed.
Camptothecin (CPT) has anticancer, antiviral, and antifungal properties. However, there is a dearth of information about antibacterial activity of CPT. Therefore, in this study, we investigated the inhibitory effect of CPT on Acidovorax avenae subsp. avenae strain RS-2, the pathogen of rice bacterial brown stripe, by measuring cell growth, DNA damage, cell membrane integrity, the expression of secretion systems, and topoisomerase-related genes, as well as the secretion of effector protein Hcp. Results indicated that CPT solutions at 0.05, 0.25, and 0.50 mg/mL inhibited the growth of strain RS-2 in vitro, while the inhibitory efficiency increased with an increase in CPT concentration, pH, and incubation time. Furthermore, CPT treatment affected bacterial growth and replication by causing membrane damage, which was evidenced by transmission electron microscopic observation and live/dead cell staining. In addition, quantitative real-time PCR analysis indicated that CPT treatment caused differential expression of eight secretion system-related genes and one topoisomerase-related gene, while the up-regulated expression of hcp could be justified by the increased secretion of Hcp based on the ELISA test. Overall, this study indicated that CPT has the potential to control the bacterial brown stripe pathogen of rice.
The progression of breast cancer is increasing at an alarming rate, particularly in western countries. Meanwhile, the lower incidence in Asian countries could be attributed to the heavy incorporation of green leaves vegetables or spices in their diets. Murraya koenigii (MK) or often times known as curry leaves are common spice used mostly in tropical countries. Anti-inflammatory and chemopreventive effects of MK aqueous extract on 4T1 breast cancer cell-challenged mice were evaluated.
Herein, cytotoxic activity of MK was first tested on 4T1 cells in vitroby MTT assay. Then, in vivo chemopreventive study was conducted where mice were fed with extracts prior to and after inducing the tumor (inoculation). Tumor size was monitored post-4T1 inoculation. At the end of experiment, histopathology of tumor sections, T cell immunophenotyping, tumor nitric oxide level, serum cytokine level and qPCR analysis on expression of iNOS, iCAM, NF-kB and c-MYC were performed.
MK reduced the tumors' size and lung metastasis aside from inhibited the viability of 4T1 cells in vitro. Furthermore, it decreased the level of nitric oxide and inflammation-related cytokines and genes, including iNOS, iCAM, NF-kB and c-MYC.
The results propose that, MK managed to inhibit the progression of tumor via immunostimulatory effect and inflammatory reaction within the tumor samples. This suggests that MKconsumption could be a savior in the search of new chemopreventive agents.
Objectives: The present investigation was carried out with an objective of formulating prototype controlled release (CR) matrix tablets of Camptothecin (CPT), an anti-cancer drug. Experimental: pH solubility profile studies of CPT and solubility of CPT in different surfactants were carried out. Controlled release tablets were prepared by direct compression method using hydroxyl propylmethyl cellulose (HPMC LVCR and HPMC K4M) as release retardants. The effect of different grades of microcrystalline cellulose (MCC) like Avicel PH 101 and Avicel PH 105 and solubilizing agents like cyclodextrins were included in the formulations and their effect on CPT release from tablets were studied. Results and Discussion: Solubility studies were conducted in order to select suitable dissolution medium for CPT. Based on the solubility studies, 0.1N HCl with 3% w/v SLS (pH1.2) was selected as dissolution medium. The FTIR and DSC results indicated that there was no in situ interaction between CPT and the selected excipients in the formualtions. Formulations containing 20% w/w of hydroxypropyl-β-cyclodextrin with Avicel PH105 as filler and HPMC k4M as release retardant gave superior CPT release of 98.40 ± 1.02% at the end of 12 h and fulfilled the regulatory requirements. The Higuchi square root model showed higher correlation coefficient values (0.949-0.990) indicating diffusion was the release mechanism for CPT from tablets. Conclusion: CPT can be formulated in to CR matrix tablets using HPMC K4M as release retardant and MCC as filler for better therapeutic efficacy.
Camptothecin (CPT), a potent antitumor drug, exhibits poor aqueous solubility and rapid conversion from the pharmacologically active lactone form to inactive carboxylate form at physiological pH. Solid dispersion of CPT in Soluplus®, an amphiphilic polymeric solubilizer, was prepared to increase the aqueous solubility of CPT and the resultant solid dispersion along with citric acid was formulated as hard gelatin capsules that were subsequently coated with Eudragit S100 polymer for colonic delivery. FTIR spectrum of the solid dispersion confirmed the presence of CPT. PXRD and DSC revealed the semicrystalline nature of solid dispersion. The solubility of the drug was found to increase ~40 times in the presence of Soluplus and ~75 times in solid dispersion. The capsules showed no drug release in 0.01 N HCl but released 86.4% drug in lactone form in phosphate buffer (pH 7.4) and the result appears to be due to citric acid-induced lowering of pH of buffer from 7.4 to 6.0. Thus the presence of citric acid in the formulation led to stabilization of the drug in its pharmacologically active lactone form. Cytotoxicity studies conducted with the formulation of solid dispersion with citric acid, utilizing cell cytotoxicity test (MTT test) on Caco-2 cells, confirmed cytotoxic nature of the formulation.
Camptothecin (CPT), a traditional anti-tumor drug, has been shown to possess anti-HIV-1 activity. To increase the antiviral potency, the anti-HIV activities of two CPT derivatives, 10-hydroxy-CPT and 7-hydroxymethyl-CPT, were evaluated in vitro. The therapy index (TI) of CPT, 10-hydroxy-CPT and 7-hydroxymethyl-CPT against HIV-1IIIB in C8166 were 24.2, 4.2 and 198.1, and against clinical isolated strain HIV-1KM018 in PBMC were 10.3, 3.5 and 66.0, respectively. While the TI of CPT, 10-hydroxy-CPT and 7-hydroxymethyl-CPT against HIV-2CBL-20 were 34.5, 10.7 and 317.0, respectively, and the TI of the three compounds against HIV-2ROD showed the similar values. However, when the antiviral mechanisms were considered, we found there was no inhibition of 7-hydroxymethyl-CPT on viral cell-to-cell transmission, and was no inhibition on reverse transcriptase, protease or integrase in cell-free systems. 7-Hydroxymethyl-CPT showed no selective killing of chronically infected cells after 3 days of incubation. In conclusion, 7-hydroxymethyl-CPT showed more potent anti-HIV activity, while 10-hydroxy-CPT had less efficient activity, compared with the parent CPT. Though the antiviral mechanisms remain to be further elucidated; the modification of -OH residues at C-7 of CPT could enhance the antiviral activity, while of -OH residues at C-10 of CPT had decreased the antiviral activity, which provides the preliminary modification strategy for anti-viral activities enhancement of this compound.
The present work describes the isolation of camptothecin and 9-methoxycamptothecin from the aerial parts of Nothapodytes foetida by semipreparative high-performance liquid chromatography because the separation of compounds by conventional procedures is tedious and cumbersome. The purity of the isolates is determined by physicochemical data and liquid chromatography-mass spectrometry.
Two new alkaloids, 9-methoxy-18,19-dehydrocamptothecin (1) and 5-hydroxymappicine-20-O-β-glucopyranoside (2a/2b as a racemic mixture), together with nine known compounds: camptothecin (3), 9-methoxy-camptothecin (4), 5-hydroxy-camptothecin (5a/5b racemic mixture), 5-hydroxy-9-methoxycamptothecin (6a/6b racemic mixture), diosmetin (7), apigenin (8), apigenin-7-O-glucopyranoside (9), rosin (cinnamyl-O-β-D-glucopyranoside) (10) and amarantholidoside IV (11) were isolated from the immature seeds of Nothapodytes foetida (Wight) Sleumer. The structures were elucidated by spectroscopic analyses. In the present research, compounds 1, 3, 4, 5a/5b and 6a/6b, also showed in vitro cytotoxicity against six cancer cell lines (HepG2, Hep3B, MDA-MB-231, MCF-7, A549, and Ca9-22). Among them, compound 1 exhibited significant cytotoxicity against these cancer cell lines, with IC50 of 0.24-6.57 μM. Furthermore, HPLC profiles were developed for qualitative and quantitative analysis of these active constituents in different parts of this plant, including mature and immature seeds, leaves, stems and roots. The results revealed that compounds 3 and 4 have the highest concentrations, which are found in the roots part of the plant.
Objectives:
Among infectious diseases, bloodstream infection (BSI) caused by gram-negative bacteria (E. coli) is the leading cause of death worldwide. However, the bacteria have produced resistance to many of these antibiotics. Thus, the present study aimed to develop silver nanoparticles (AgNPs) loaded with Emilia Sonchifolia (ES) extract (ES-AgNPs) to treat BSI efficiently.
Methods:
AgNPs were synthesized by reduction of silver nitrate (AgNO3) solution by ES extract. Furthermore, these ES-AgNPs were characterized for particle size and zeta potential, crystallinity by powder X-ray diffraction (P-XRD) technique, in vitro antibacterial activity, time-kill assay, film bio adhesion, and fluorescence assay.
Results:
Surface plasmon resonance (SPR) has been used to confirm the formation of AgNPs by seeing a shift in colour to dark-brown. The ES-AgNPs displayed a mean particle size of 137±3 nm (PDI of 0.168±0.02) and zeta potential of 18.2±0.8 mV. Furthermore, according to P-XRD results, the developed AgNPs are highly crystalline. The ES-AgNPs showed effective antibacterial action against E. coli with minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of 0.4±0.02μg/mL and 0.8±0.03μg/mL, respectively. In addition, ES-AgNPs inhibited biofilm formation and bacterial adhesion in a dose-dependent manner with 100% inhibition obtained in 48h at MBC.
Conclusions:
Present research work revealed that the ES-AgNPs obtained by green synthesis holds a prominent antibacterial activity in the treatment of BSIs caused by E. coli and they may be used as a competent substitute for current treatments. However, further, in vivo antibacterial studies are required to establish its efficacy in the treatment of BSIs.
Aim of the present investigation is to develop a curcumin loaded nanocochleates (CU-NC) from the preformed nanoliposomes (CU-NL) using a design of experiments. Plackett–Burman screening design (PBD) was implemented to investigate the effects of formulation and process variables on particle size (Y1) and Entrapment Efficiency (Y2). Stirring speed (X5), amount of phospholipid (X4) and cholesterol (X8) were identified as significant (P < 0.05) factors, which were further optimized using Box-Behnken design (BBD). Optimized CU-NC showed particle size, Entrapment efficiency and zeta potential of 204.14 nm, 84.21 ± 1.91 and - 8.20 mV respectively. Curcumin was partially crystalline and amorphous form in Cu-NL and CU-NC respectively, X-ray diffraction and thermal study revealed. CU-NC retarded drug release up to 24 h and followed Higuchi release kinetic model. Noteworthy CU-NC showed 45.77 and 85.81% reduction in breast cancer MCF-7 cells viability than CU-NL and curcumin dispersion respectively. CU-NC treated cells showed nuclear condensation, and nuclear fragmentation of cells and the apoptotic bodies when observed using cell permeable nucleic acid stain. Statistical design of experiments can be used to formulate the CU-NC at the optimized conditions showing the desired constraints for particle size and entrapment efficiency with better anticancer potential.
Camptothecin (CPT) was discovered from plant extracts more than 60 years ago. Since then, only two CPT analogues (irinotecan and topotecan) have been approved for cancer treatment, although several thousand CPT derivatives have been synthesized and many of them were actively studied in our research community over the past 6+ decades. In this review article, we briefly summarize: (1) the discovery and early development of CPTs, (2) the recognized CPT mechanism of action (MOA), (3) the synthesis of CPT and CPT analogues, and (4) the structureactivity relationship (SAR) of CPT and its analogues. Next, we provide evidence that certain CPT analogues can exert improved efficacy with low toxicity independently of topoisomerase I (Top1) inhibition; instead, these CPT analogues use novel MOAs by targeting important cancer survival-associated oncogenic proteins and/or by bypassing various treatment-resistant mechanisms. We then present a comprehensive review of the most advanced CPT analogues in clinical development, with the goal of resolving why no new CPTs have been FDA approved for cancer treatment, beyond irinotecan and topotecan. We argue that new CPT Top1 inhibitor drugs are unlikely being found to be significantly better than irinotecan and/or topotecan in terms of the overall antitumor activity and toxicity. The significance of CPT analogues that possess novel MOAs has not been sufficiently recognized so far. In our opinion, this is a research area with great potential to make a breakthrough for development of the next generation of CPT analogues that possess high efficacy (due to novel targets) and low toxicity (due to low inhibition of Top1 activity/function) for effective treatment of human disease, including cancer.
DNA topoisomerases represent an essential family of DNA processing enzymes and a large number of topoisomerase inhibitors are used clinically for the treatment of various human cancers. Novels drugs are in clinical development both against type I and type II topoisomerases. The book will include basic biochemical and structural reviews for the cancer-relevant topoisomerases. It will describe how topoisomerase dysfunctions can damage the genome and increase the risk of cancers, and the involvement of topoisomerases in programmed cell death. The book will also present the various topoisomerase inhibitors in clinical use and development and their molecular and cellular mechanisms of action. © Springer Science+Business Media, LLC 2012. All rights reserved.
Enterovirus 71 (EV71) is one of the causative agents of hand, foot and mouth disease (HFMD) associated with severe neurological disease. EV71's pathogenesis remains poorly understood and the lack of approved antiviral has led to its emergence as a clinically important neurotropic virus. The goals of this study were to: (i) identify novel anti-EV71 compounds that may serve as lead molecules for therapeutics; and (ii) investigate their targets in downstream studies. We screened a 502-compound library of highly purified natural products for anti-EV71 activities in a cell-based immunofluorescence assay that were then confirmed in viral plaque reduction assays. Along with known antivirals, novel inhibitors of EV71 were also identified. We selected camptothecin for downstream studies and found that it is a limited spectrum enterovirus inhibitor that inhibits coxsackievirus A16 but not ECHOvirus 7. Camptothecin, a DNA topoisomerase 1 (TOP1) inhibitor, inhibits both viral RNA replication and translation based on luciferase replicon studies. Depletion of TOP1 using siRNA was then able to rescue EV71 infection from camptothecin inhibition. Interestingly, EV71 viral RNA replication and translation were also in TOP1 depleted cells. We found that nuclear TOP1 was relocalized to cytoplasmic replication vesicles during EV71 infection and localized with viral 3CD using confocal microscopy and proximity-ligation assays. Our findings reveal camptothecin to be a limited spectrum antiviral against enteroviruses that functions in a TOP1-dependent but cytotoxicity-independent manner. TOP1 is in turn needed for maximal EV71 viral RNA replication and viral protein synthesis.
Cancer is the second leading cause of death worldwide behind cardiovascular disease (1). According to the Global Burden of Disease (GBD) 2015 Study, which was conducted as part of a comprehensive regional and global research program assessing the mortality and disability caused by major diseases, injuries, and risk factors and involving over 500 researchers representing over 300 institutions and 50 countries, cancer mortality decreased between 2005 and 2015 despite the global incidence rates of cancer increasing during this period (1).
PURPOSE: The combination of chemotherapy with immunotherapeutic agents such as interleukin-2 and interferon alfa-2b has been reported to provide improved treatment results in patients with metastatic melanoma, compared with the use of chemotherapy alone. We have performed a prospective randomized trial in patients with metastatic melanoma, comparing treatment with chemotherapy to treatment with chemoimmunotherapy.
PATIENTS AND METHODS: One hundred two patients with metastatic melanoma were prospectively randomized to receive chemotherapy composed of tamoxifen, cisplatin, and dacarbazine or this same chemotherapy followed by interferon alfa-2b and interleukin-2. Objective responses, survival, and toxicity in the two groups were evaluated at a median potential follow-up of 42 months.
RESULTS: In 52 patients randomized to receive chemotherapy, there were 14 objective responses (27%), including four complete responses. In 50 patients randomized to receive chemoimmunotherapy, there were 22 objective responses (44%) (P2 = .071), including three complete responses. In both treatment groups, the duration of partial responses was often short, and there was a trend toward a survival advantage for patients receiving chemotherapy alone (P2 = .052; median survival of 15.8 months compared with 10.7 months). Treatment-related toxicities were greater in patients receiving chemoimmunotherapy.
CONCLUSION: With the treatment regimens used in this study, the addition of immunotherapy to combination chemotherapy increased toxicity but did not increase survival. The use of combination chemoimmunotherapy regimens is not recommended in the absence of well-designed, prospective, randomized protocols showing the benefit of this treatment strategy.
A rapid and sensitive liquid chromatography-tandem mass spectrometry method with multiple reaction monitoring was developed for the simultaneous determination of camptothecin and 10-hydroxycamptothecin in Camptotheca acuminata. The separation of camptothecin and 10-hydroxycamptothecin was performed on an Agilent Eclipse XDB-C18 column with a mixture of methanol and water (1:1, v/v) containing 0.2% formic acid as a mobile phase. The limits of detection of camptothecin and 10-hydroxycamptothecin were 4.0 ng/mL and 7.0 ng/mL (S/N = 3), respectively. Analysis took 10 minutes, making the method suitable for rapid determination of camptothecin and 10-hydroxycamptothecin in C. acuminata.
Topoisomerases are ubiquitous enzymes that control DNA supercoiling and entanglements. They are essential during transcription and replication and topoisomerase inhibitors are among the most effective and most commonly used anticancer and antibacterial drugs. This review consists in two parts. In the first part ("Lessons"), it gives background information on the catalytic mechanisms of the different enzyme families (6 different genes in humans and 4 in most bacteria), describes the "interfacial inhibition" by which topoisomerase-targeted drugs act as topoisomerase poisons and describes clinically relevant topoisomerase inhibitors. It generalizes the interfacial inhibition principle, which was discovered from the mechanism of action of topoisomerase inhibitors, and discusses how topoisomerase inhibitors kill cells by trapping topoisomerases on DNA rather than by classical enzymatic inhibition. Trapping protein-DNA complexes extends to a novel mechanism of action of PARP inhibitors and could be applied to the targeting of transcription factors. The second part of the review focuses on the challenges for discovery and precise use of topoisomerase inhibitors, including targeting topoisomerase inhibitors using chemical coupling and encapsulation for selective tumor delivery, use of pharmacodynamic biomarkers to follow drug activity, complexity of the response determinants for anticancer activity and patient selection, prospects of rational combinations with DNA repair inhibitors targeting tyrosyl-DNA-phosphodiesterases 1 and 2 (TDP1 and TDP2) and PARP, and the unmeet need to develop inhibitors for type IA enzymes.
Metastatic melanoma is commonly treated with chemotherapy and/or biological agents used separately. In this study we have investigated the efficacy of combined chemotherapy using cisplatin, vinblastine, DTIC (CVD) and biological therapy using interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) in patients with metastatic melanoma.
All patients had advanced, inoperable melanoma without prior treatment with chemotherapy or biotherapy, a performance status of ECOG 0-2 and no evidence of symptomatic brain metastases. The CVD regimen consisted of cisplatin 20 mg/m2/d x 4, vinblastine 1.6 mg/m2/d x 5 and DTIC 800 mg/m2 x 1, repeated at 21-day intervals. The biotherapy regimen included IL-2, 9 x 10(6) IU/ m2/d x 4 days and IFN-alpha 5 x 10(6) U/m2/d SC x 5 days. The CVD and biotherapy regimens were integrated initially, in an alternating manner at 6-week intervals and subsequently, in a sequential fashion where patients were randomized to receive either CVD immediately followed by biotherapy (CVD/Bio) or the reverse sequence (Bio/CVD). Patients were admitted to the hospital for IL-2 administration and for monitoring and treatment of IL-2 induced side effects. The phase II results of the integrated therapy (biochemotherapy) studies were retrospectively compared to our previously reported results with the CVD regimen used alone.
The alternating biochemotherapy program was used in 40 patients and the sequential biochemotherapy was used in 62 patients. The alternating regimen produced 2 CRs and 11 PRs for an overall response rate of 33% among 39 evaluable patients. The sequential biochemotherapy produced 14 CRs and 23 PRs for an overall response rate of 60% (95% CI, 47% to 72%). The sequence of CVD/Bio resulted in a higher response rate (11 CRs + 11 PRs (69%)) compared to the Bio/CVD sequence (3 CRs + 12 PRs (50%)). Although the duration of PRs was short (median, 8 months), the median duration of CRs was 3+years and 10 of 16 CRs are currently disease free for periods of 3+ to 6+ years. The median survival of patients receiving sequential biochemotherapy was 13 months compared to 9 months for the CVD treated group (P = 0.04). Treatment with biochemotherapy was associated with severe toxicity including intense myelosuppression, infections, IL-2 induced constitutional toxicity and hypotension. However, the IL-2 induced toxicities were generally manageable on a regular ward, except for 15% of the patients who required transfer to an intensive care unit for treatment of complications associated with the treatment.
The sequential combination of CVD with IL-2 + IFN-alpha appears to have produced an increase in the number of durable responses in patients with metastatic melanoma. The toxicity of this program, although severe, was manageable. The biochemotherapy regimen produced an apparent increase in the median survival compared to that observed with the CVD regimen. However, a prospective comparison of these two regimens will be required to confirm these observations.
Camptothecin (CPT) class of compounds has been demonstrated to be effective against a broad spectrum of tumors. Their molecular target has been firmly established to be human DNA topoisomerase I (topo I). CPT inhibits topo I by blocking the rejoining step of the cleavage/religation reaction of topo-I, resulting in accumulation of a covalent reaction intermediate, the cleavable complex. The primary mechanism of cell killing by CPT is S-phase-specific killing through potentially lethal collisions between advancing replication forks and topo-I cleavable complexes. Collisions with the transcription machinery have also been shown to trigger the formation of long-lived covalent topo-I DNA complexes, which contribute to CPT cytotoxicity. Two novel repair responses to topo-I-mediated DNA damage involving covalent modifications of topo-I have been discovered. The first involves activation of the ubiquitin/26S proteasome pathway, leading to degradation of topo-I (CPT-induced topo-I downregulation). The second involves SUMO conjugation to topo-I. The potentials roles of these new mechanisms for repair of topo-I-mediated DNA damage in determining CPT sensitivity/resistance in tumor cells are discussed.
A quantitative analysis using (1)H-NMR has been developed for the determination of camptothecin derivatives and trigonelline in Nothapodytes foetida root, stems and leaves. In the region of delta 9.5-5.5, the signals of H-7 of camptothecin (1), H-10 of 9-methoxycamptothecin (2), H-19 of pumiloside (3) and H-2 of trigonelline (4), were well separated from each other in DMSO-d(6). The quantity of the compounds was calculated by the ratio of the intensity of each compound to the known amount of internal standard 3,4,5-trimethoxybenzaldehyde. These results were compared with the conventional HPLC method. The advantages of the method are that no reference compounds are required for calibration curves, the quantification could be directly realized on a crude extract, an overall profile of the preparation could be directly obtained, and a very significant time-gain could be achieved, in comparison to conventional HPLC methods, for instance.
Nuclear DNA topoisomerase I (TOP1) is an essential human enzyme. It is the only known target of the alkaloid camptothecin, from which the potent anticancer agents irinotecan and topotecan are derived. As camptothecins bind at the interface of the TOP1-DNA complex, they represent a paradigm for interfacial inhibitors that reversibly trap macromolecular complexes. Several camptothecin and non-camptothecin derivatives are being developed to further increase anti-tumour activity and reduce side effects. The mechanisms and molecular determinants of tumour response to TOP1 inhibitors are reviewed, and rational combinations of TOP1 inhibitors with other drugs are considered based on current knowledge of repair and checkpoint pathways that are associated with TOP1-mediated DNA damage.
Camptothecin (CPT), a monoterpene alkaloid, is an important anti-cancer compound obtained from several plant sources including Camptotheca acuminta (from China) and Nothapodytes nimmoniana (from India). Currently, by far the highest levels of CPT (approximately 0.3% w/w) are reported from Nothapodytes nimmoniana, a small tree distributed in the Western Ghats, India. In recent years because of the heavy demand, there has been a serious threat of extinction of the populations of the tree in the Western Ghats forest of south India. Several studies have chemically profiled populations of the species in the Western Ghats to identify sources of high yield and therefore to enable the sustainable production and harvesting of CPT. In this study, using both high-performance liquid chromatography and liquid chromatography-mass spectrometry, we report for the first time the identification of trees that produce at least 5- to 8-fold more CPT than hitherto reported. Furthermore, we show for the first time the production of a few minor camptothecines, including 10-hydroxy camptothecin, in the stem and root bark extracts of the tree. These results have important implications for not only harnessing the high-yielding individuals for clonal multiplication but also for exploiting some of the minor camptothecines, which also have been shown to have important anti-cancer and anti-viral activity.
Formulation development and standardization of herbal gel containing methanolic extract of Butea frontosa
- U Mishra
- P N Murthy
- G Pasa
- S Kumar
HPLC Confirmation of Camptothecin an Anticancer Metabolite from Bark Extract of Nothapodytes nimmoniana
- A Patil
- S Patil
- Uv
- Ftir
Analysis of Nothapodytes nimmoniana [J. Graham] mabberly leaves
- V Balasubramaniam
- Gc-Ms