ETHNOPHARMACOLOGICAL RELEVANCE: Annona muricata known as "the cancer killer" has been widely used in the traditional medicine for the treatment of cancer and tumors. The purpose of this study is to investigate the anticancer properties of ethyl acetate extract of Annona muricata leaves (EEAM) on HT-29 and HCT-116 colon cancer cells and the underlying mechanisms.
MATERIALS AND METHODS: The effect of EEAM on the cell proliferation of HT-29 and HCT-116 cells was analyzed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay. High content screening system (HCS) was applied to investigate the cell membrane permeability, mitochondrial membrane potential (MMP), nuclear condensation and cytochrome c translocation from mitochondria to cytosol. Reactive oxygen species (ROS) formation, lactate dehydrogenase (LDH) release and activation of caspase-3/7, -8 and -9 were measured while treatment. Flow cytometric analysis was used to determine the cell cycle distribution and phosphatidylserine externalization. The protein expression of Bax and Bcl-2 was determined using immunofluorescence analysis. In addition, the potential of EEAM to suppress the migration and invasion of colon cancer cells was also examined.
RESULTS: EEAM exerted significant cytotoxic effects on HCT-116 and HT-29 cells as determined by MTT and LDH assays. After 24 h treatment, EEAM exhibited the IC₅₀ value of 11.43 ± 1.87 µg/ml and 8.98 ± 1.24 µg/ml against HT-29 and HCT-116 cells, respectively. Flow cytometric analysis demonstrated the cell cycle arrest at G1 phase and phosphatidylserine externalization confirming the induction of apoptosis. EEAM treatment caused excessive accumulation of ROS followed by disruption of MMP, cytochrome c leakage and activation of the initiator and executioner caspases in both colon cancer cells. Immunofluorescence analysis depicted the up-regulation of Bax and down-regulation of Bcl-2 proteins while treated with EEAM. Furthermore, EEAM conspicuously blocked the migration and invasion of HT-29 and HCT-116 cells.
CONCLUSIONS: These findings provide a scientific basis for the use of A. muricata leaves in the treatment of cancer, although further in vivo studies are still required.
Nanomedicine is a rapidly emerging field and is reported to be a promising tool for treating various diseases. Green synthesized nanoparticles are documented to possess a potent anticancer effect. Rabdosia rubescens is a Chinese plant which is also one of the components of PC-SPES and used to treat prostate cancer. In the present study, we synthesized the gold nanoparticles from R. rubescens (RR-AuNP) and analyzed its anticancer activity against the lung carcinoma A549 cell lines. Since lung cancer is reported to be with increased morbidity and decreased survival rate. The biosynthesized RR-AuNP were confirmed using UV-Visible spectrophotometer, size and shape of RR-AuNP were assessed by DLS, TEM and EDX. The biomolecules present in RR-AuNP and its topographical structure were detected using FTIR, SAED and AFM analysis. MTT assay was performed to detect the IC50 dose of RR-AuNP and its apoptotic effect was assessed by detecting the caspases activation, ROS generation. The anticancer effect of RR-AuNP was confirmed by DAPI staining, TUNEL assay and its molecular mechanism were confirmed by assessing the apoptotic signalling molecules protein expression. Our results illustrate that RR-AuNP showed a strong absorption peak at 550 nm and the RRAuNP were polydispersed nanospheres with size of 130 nm. RR-AuNP IC50 dose against A549 lung carcinoma cell line was detected to be at 25 µg/ml. The results of DAPI staining, TUNEL and immunoblotting analysis confirms both the 25 µg/ml and 50 µg/ml of RR-AuNP possess potent anticancer and apoptotic effect, suggesting that RR-AuNP that it may be a persuasive molecule to treat lung cancer.
The asymptomatic properties and high treatment resistance of ovarian cancer result in poor treatment outcomes and high mortality rates. Although the fundamental chemotherapy provides promising anticancer activities, it is associated with severe side effects. The derivative of phenothiazine, namely, 10H-3,6-diazaphenothiazine (PTZ), was synthesized and reported with ideal anticancer effects in a previous paper. In this study, detailed anticancer properties of PTZ was examined on A2780 ovarian cancer cells by investigating the cytotoxicity profiles, mechanism of apoptosis, and cell invasion. Research outcomes revealed PTZ-induced dose-dependent inhibition on A2780 cancer cells (IC50 =0.62 µM), with significant less cytotoxicity toward HEK293 normal kidney cells and H9C2 normal heart cells. Generation of reactive oxygen species (ROS) and polarization of mitochondrial membrane potential (ΔΨm) suggests PTZ-induced cell death through oxidative damage. The RT2 Profiler PCR Array on apoptosis pathway demonstrated PTZ-induced apoptosis via intrinsic (mitochondria-dependent) and extrinsic (cell death receptor-dependent) pathway. Inhibition of NF-κB and subsequent inhibition of (BIRC6-XIAP) complex activities reduced the invasion rate of A2780 cancer cells penetrating through the Matrigel™ Invasion Chamber. Lastly, the cell cycle analysis hypothesizes that the compound is cytostatic and significantly arrests cell proliferation at G2/M phase. Hence, the exploration of the underlying anticancer mechanism of PTZ suggested its usage as promising chemotherapeutic agent.
The anti-carcinogenic effect of the new quinazolinone compound, named MMD, was tested on MCF-7 human breast cancer cell line. The synthesis of quinazolinone-based compounds attracted strong attention over the past few decades as an alternative mean to produce analogues of natural products. Quinazolinone compounds sharing the main principal core structures are currently introduced in the clinical trials and pharmaceutical markets as anti-cancer agents. Thus, it is of high clinical interest to identify a new drug that could be used to control the growth and expansion of cancer cells. Quinazolinone is a metabolite derivative resulting from the conjugation of 2-aminobenzoyhydrazide and 5-methoxy-2- hydroxybenzaldehyde based on condensation reactions. In the present study, we analysed the influence of MMD on breast cancer adenoma cell morphology, cell cycle arrest, DNA fragmentation, cytochrome c release and caspases activity. MCF-7 is a type of cell line representing the breast cancer adenoma cells that can be expanded and differentiated in culture. Using different in vitro strategies and specific antibodies, we demonstrate a novel role for MMD in the inhibition of cell proliferation and initiation of the programmed cell death. MMD was found to increase cytochrome c release from the mitochondria to the cytosol and this effect was enhanced over time with effective IC50 value of 5.85 ± 0.71 μg/mL detected in a 72-hours treatment. Additionally, MMD induced cell cycle arrest at G0/G1 phase and caused DNA fragmentation with obvious activation of caspase-9 and caspases-3/7. Our results demonstrate a novel role of MMD as an anti-proliferative agent and imply the involvement of mitochondrial intrinsic pathway in the observed apoptosis.
The leucine aminopeptidase inhibitor, benzyloxycarbonyl-leucine-chloromethylketone (z-L-CMK), was found to be toxic and readily induce cell death in Jurkat T cells. Dose-response studies show that lower concentration of z-L-CMK induced apoptosis in Jurkat T cells whereas higher concentration causes necrosis. In z-L-CMK-induced apoptosis, both the initiator caspases (-8 and -9) and effector caspases (-3 and -6) were processed to their respective subunits. However, the caspases remained intact in z-L-CMK-induced necrosis. The caspase inhibitor, z-VAD-FMK inhibited z-L-CMK-mediated apoptosis and caspase processing but has no effect on z-L-CMK-induced necrosis in Jurkat T cells. The high mobility group protein B1 (HMGB1) protein was found to be released into the culture medium by the necrotic cells and not the apoptotic cells. These results indicate that the necrotic cell death mediated by z-L-CMK at high concentrations is via classical necrosis rather than secondary necrosis. We also demonstrated that cell death mediated by z-L-CMK was associated with oxidative stress via the depletion of intracellular glutathione (GSH) and increase in reactive oxygen species (ROS), which was blocked by N-acetyl cysteine. Taken together, the results demonstrated that z-L-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. The toxic side effects in Jurkat T cells mediated by z-L-CMK are associated with oxidative stress via the depletion of GSH and accumulation of ROS.
Tioman virus is a newly described bat-urine derived paramyxovirus isolated in Tioman Island, Malaysia in 2001. Hitherto, neither human nor animal infection by this virus has been reported. Nonetheless, its close relationship to another paramyxovirus, the Menangle virus which had caused diseases in humans and pigs [Philbey, A.W., Kirkland, P.D., Ross, A.D., Davis, R.J., Gleeson, A.B., Love, R.J., Daniels, P.W., Gould, A.R., Hyatt, A.D., 1998. An apparently new virus (family Paramyxoviridae) infectious for pigs, humans, and fruit bats. Emerg. Infect. Dis. 4, 269-271], raises the possibility that it may be potentially pathogenic. In this study, mice were experimentally infected with Tioman virus by intraperitoneal and intracerebral routes, and the cellular targets and topographical distribution of viral genome and antigens were examined using in situ hybridization and immunohistochemistry, respectively. The possible association between viral infection and apoptosis was also investigated using the TUNEL assay and immunohistochemistry to FasL, Caspase-3, Caspase-8, Caspase-9 and bcl-2. The results showed that Tioman virus inoculated intracerebrally was neurotropic causing plaque-like necrotic areas, and appeared to preferentially replicate in the neocortex and limbic system. Viral infection of inflammatory cells was also demonstrated. TUNEL and Caspase-3 positivity was found in inflammatory cells but not in neurons, while FasL, Caspase-8 and Caspase-9 were consistently negative. This suggests that neuronal infection was associated with necrosis rather than apoptosis. Moreover, the data suggest that there may be an association between viral infection and apoptosis in inflammatory cells, and that it could, at least in part, involve Caspase-independent pathways. Bcl-2 was expressed in some neurons and inflammatory cells indicating its possible role in anti-apoptosis. There was no evidence of central nervous system infection via the intraperitoneal route.
Cancer has become the primary cause of death worldwide, and anticancer drugs are used to combat this disease. Synthesis of anticancer drugs has limited success due to adverse side effects has made compounds from natural products with minimal toxicity gain much popularity. Piper species are known to have a biological effect on human health. The biological activity is due to Piper species rich with active secondary metabolites that can combat most diseases, including cancer. This review will discuss the phytochemistry of Piper species and their anticancer activity. The identification and characterization of ten active metabolites isolated from Piper species were discussed in detail and their anticancer mechanism. These metabolites were mainly found could inhibit anticancer through caspase and P38/JNK pathways. The findings discussed in this review support the therapeutic potential of Piper species against cancer due to their rich source of active metabolites with demonstrated anticancer activity.
BACKGROUND: Clausena excavata Burm.f. is a shrub traditionally used to treat cancer patients in Asia. The main bioactive chemical components of the plant are alkaloids and coumarins. In this study, we isolated clausenidin from the roots of C. excavata to determine its apoptotic effect on the colon cancer (HT-29) cell line.
METHOD: We examined the effect of clausenidin on cell viability, ROS generation, DNA fragmentation, mitochondrial membrane potential in HT-29 cells. Ultrastructural analysis was conducted for morphological evidence of apoptosis in the treated HT-29 cells. In addition, we also evaluated the effect of clausenidin treatment on the expression of caspase 3 and 9 genes and proteins in HT-29 cells.
RESULT: Clausenidin induced a G0/G1 cell cycle arrest in HT-29 cells with significant (p
Dillenia suffruticosa, which is locally known as Simpoh air, has been traditionally used to treat cancerous growth. The ethyl acetate extract of D. suffruticosa (EADs) has been shown to induce apoptosis in MCF-7 breast cancer cells in our previous study. The present study aimed to elucidate the molecular mechanisms involved in EADs-induced apoptosis and to identify the major compounds in the extract. EADs was found to promote oxidative stress in MCF-7 cells that led to cell death because the pre-treatment with antioxidants α-tocopherol and ascorbic acid significantly reduced the cytotoxicity of the extract (P<0.05). DCFH-DA assay revealed that treatment with EADs attenuated the generation of intracellular ROS. Apoptosis induced by EADs was not inhibited by the use of caspase-inhibitor Z-VAD-FMK, suggesting that the cell death is caspase-independent. The use of JC-1 dye reflected that EADs caused disruption in the mitochondrial membrane potential. The related molecular pathways involved in EADs-induced apoptosis were determined by GeXP multiplex system and Western blot analysis. EADs is postulated to induce cell cycle arrest that is p53- and p21-dependent based on the upregulated expression of p53 and p21 (P<0.05). The expression of Bax was upregulated with downregulation of Bcl-2 following treatment with EADs. The elevated Bax/Bcl-2 ratio and the depolarization of mitochondrial membrane potential suggest that EADs-induced apoptosis is mitochondria-dependent. The expression of oxidative stress-related AKT, p-AKT, ERK, and p-ERK was downregulated with upregulation of JNK and p-JNK. The data indicate that induction of oxidative-stress related apoptosis by EADs was mediated by inhibition of AKT and ERK, and activation of JNK. The isolation of compounds in EADs was carried out using column chromatography and elucidated using the nuclear resonance magnetic analysis producing a total of six compounds including 3-epimaslinic acid, kaempferol, kaempferide, protocatechuic acid, gallic acid and β-sitosterol-3-O-β-D-glucopyranoside. The cytotoxicity of the isolated compounds was determined using MTT assay. Gallic acid was found to be most cytotoxic against MCF-7 cell line compared to others, with IC50 of 36 ± 1.7 μg/mL (P<0.05). In summary, EADs generated oxidative stress, induced cell cycle arrest and apoptosis in MCF-7 cells by regulating numerous genes and proteins that are involved in the apoptotic signal transduction pathway. Therefore, EADs has the potential to be developed as an anti-cancer agent against breast cancer.
Besides acting as potent free radical scavengers, tocopherols and tocotrienols have been known to have non-antioxidant properties such as the involvement of alpha-tocopherol (alphaT) in PKC pathway and the anti-cancer properties of gamma-tocotrienol (gammaT3). This study aims to elucidate whether protective effects shown by alphaT and gammaT3 in H(2)O(2)-induced neuron cultures have anti-apoptotic or pro-apoptotic tendency toward the initiation of neuronal apoptosis. H(2)O(2) is used to induce apoptosis in primary cerebellar neuron cultures which is attenuated by pretreatment of alphaT or gammaT3 at concentrations < or =10 microM. Similar to our previous work, gammaT3 was found to be neurotoxic at concentrations > or =100 microM, whereas alphaT showed no neurotoxicity. Cellular uptake of gammaT3 was higher than that of alphaT. Treating cells simultaneously with either gammaT3 or alphaT and with then H(2)O(2) led to higher expression of Bax and Bcl-2 than in neurons exposed to H(2)O(2) alone. Analysis of Bcl-2/Bax ratio as 'survival index' showed that both pretreatment of gammaT3 and alphaT followed by H(2)O(2) increase the 'survival index' of Bcl-2/Bax ratio compared to H(2)O(2)-treated cells, while treatment of gammaT3 alone decrease the ratio compared to unchanged Bcl2/Bax ratio of similar treatment with alphaT alone. Similar treatment of gammaT3 decreased p53 expression and activates p38 MAPK phosphorylation, whereas alphaT did not alter its expression compared to H(2)O(2)-treated cells. Treating neurons with only gammaT3 or alphaT increased the expression of Bax, Bcl-2, p53, and p38 MAPK compared to control with gammaT3 exerting stronger expression for proteins involved than alphaT. In conclusion, low doses of gammaT3 and alphaT confer neuroprotection to H(2)O(2)-treated neurons via their antioxidant mechanism but gammaT3 has stronger pro-apoptosis tendency than alphaT by activating molecules involved in the neuronal apoptotic pathway in the absence of H(2)O(2).
Phyla nodiflora L. has been used as medicinal remedies for various ailments due to its antioxidant, anti-inflammatory, anti-bacterial, anti-tumor activity. Previously, we found that the plant extracts induced DNA fragmentation in MCF-7. This study was to investigate the modes of action of P. nodiflora in inhibiting breast cancer cells using leaf ethyl acetate (EA leaf), stem ethyl acetate (EA stem) and stem methanol (Met stem) extracts. The MTT assay showed that the anti-proliferative effects of P. nodiflora extracts were selective towards MCF-7 with a minimal effect on MCF10A. Morphological changes such as cell shrinkage and nuclear condensation were observed in treated cells. We found that induction of apoptosis by EA leaf and EA stem was mitochondrial-dependent while loss of mitochondrial membrane potential was not found in Met stem-treated cells. In addition, the expression levels of AIFM1, CASP9, CFLAR, and IGF1R were altered after treatment. Decreased BCL-2 expression was found in treated cells while BAX and caspases' expression was upregulated or maintained. All extracts caused perturbation of cell cycle at S phase by dysregulating the expression of cell cycle regulators such as CDKs and cyclins. Our findings indicate that P. nodiflora inhibits MCF-7 cells by inducing apoptosis and perturbing cell cycle.
INTRODUCTION: This study focused on PF4 effects on caspase-3,-6, -7, -8 and -9 which regulate the apopotosis process in breast cancer.
MATERIALS AND METHODS: Breast tumours were induced in forty 21-day-old female Sprague Dawley rats (SDRs) using MNU until tumour size reached 14.5 mm (SD: 0.5 mm). The rats were then divided into two groups: Group 1 (control injected with 0.9% saline; n = 20), and Group 2 (platelet factor 4 (PF4); n = 20). PF4 was administered through focal intralesional injection at 20 μg/lesion dose. Following 5-day treatment, the SDRs were sacrificed. Subsequently, representative sections from the tumour were obtained for haematoxylin and eosin (H&E) staining. The expressions of caspase-3, -6, -7, -8 and -9 were evaluated using immunohistochemistry (IHC) staining.
RESULTS: The majority of breast tumour specimens were of aggressive types [ncontrol = 13 (65%); nPF4 = 12 (60%)]. Invasive ductal carcinoma not otherwise specified (IDC-NOS) was the most commonly observed breast tumour histology for control and PF4 groups (n = 8 (40%) in respective groups). PF4-treated group exhibited significant differences in the caspase-3, -6 and -8 expression levels compared to the control group (all p < 0.001). There were no significant differences in caspase-7 (p = 0.347) and caspase-9 (p = 0.373) expression levels between both groups.
CONCLUSION: This study found that PF4 acts via the caspase-mediated extrinsic apoptosis pathway without the involvement of the intrinsic pathway.
In the solid state each of three binuclear zinc dithiocarbamates bearing hydroxyethyl groups, {Zn[S2CN(R)CH2CH2OH]2}2 for R = iPr (1), CH2CH2OH (2), and Me (3), and an all alkyl species, [Zn(S2CNEt2)2]2 (4), features a centrosymmetric {ZnSCS}2 core with a step topology; both 1 and 3 were isolated as monohydrates. All compounds were broadly cytotoxic, specifically against human cancer cell lines compared with normal cells, with greater potency than cisplatin. Notably, some selectivity were indicated with 2 being the most potent against human ovarian carcinoma cells (cisA2780), and 4 being more cytotoxic toward multidrug resistant human breast carcinoma cells (MCF-7R), human colon adenocarcinoma cells (HT-29), and human lung adenocarcinoma epithelial cells (A549). Based on human apoptosis PCR-array analysis, caspase activities, DNA fragmentation, cell apoptotic assays, intracellular reactive oxygen species (ROS) measurements and human topoisomerase I inhibition, induction of apoptosis in HT-29 cells is demonstrated via both extrinsic and intrinsic pathways. Compounds 2-4 activate the p53 gene while 1 activates both p53 and p73. Cell cycle arrest at the S and G2/M phases correlates with inhibition of HT-29 cell growth. Cell invasion is also inhibited by 1-4 which is correlated with down-regulation of NF-κB.
Epipremnum pinnatum (L.) Engl. hexane extract produced a significant growth inhibition against T-47D breast carcinoma cells and analysis of cell death mechanisms indicated that the extract elicited a non-apoptotic programmed cell death. T-47D cells exposed to the extract at EC(50) concentration (72 h) for 24 h failed to demonstrate typical DNA fragmentation associated with apoptosis, as carried out using a modified TUNEL assay. In addition, acute exposure to the extract produced an insignificant regulation of caspase-3 and p53 mRNA expression but increased in the c-myc mRNA expression. Ultrastructural analysis using transmission electron microscope demonstrated distinct vacuolated cells, which strongly indicated a Type II non-apoptotic cell death although the changes in chromatin were also detected. The presence of non-apoptotic programmed cell death was then reconfirmed with annexin-V and propidium iodide staining. These findings suggested that up-regulation of c-myc mRNA expression may have contributed to the growth arrest and Type II non-apoptotic programmed cell death in the Epipremnum pinnatum (L.) Engl. hexane extract-treated T-47D cells.
t(11;18)(q21;q21) Translocation and trisomy 3 are the most common chromosomal aberrations reported in low-grade mucosa-associated lymphoid tissue (MALT) lymphoma. The current study aims to investigate the frequency of these chromosomal aberrations in a series of 52 extranodal B-cell lymphomas. The tumours were categorised into three histological grades: grade 1 (low-grade lymphoma of MALT type), grade 2 [diffuse large B-cell lymphoma (DLBCL) with MALT component] and grade 3 (DLBCL without MALT component). Fluorescence in situ hybridisation analyses on paraffin tissue sections were performed using a locus-specific probe for the 18q21 region and a centromeric probe for chromosome 3. The 18q21 rearrangement was detected in 9 of 40 (23%) cases, including 7 of 23 (30%) grade-1 and 2 of 11 (18%) grade-3 tumours. Amplification of the 18q21 region was detected in 10 of 40 (25%) cases, and trisomy 3 was detected in 9 of 34 (26%) cases. Amplification of the 18q21 region may be an important alternative pathogenetic pathway in MALT lymphoma and was found almost exclusively in tumours without 18q21 rearrangement. Our study showed that tumours with 18q21 rearrangement and 18q21 amplification develop along two distinct pathways, and the latter was more likely to transform into high-grade tumours upon acquisition of additional genetic alterations, such as trisomy 3. Trisomy 3 was more frequently found in coexistence with 18q21 abnormalities, suggesting that it was more likely to be a secondary aberration.
Phytochemicals from Pseuduvaria species have been reported to display a wide range of biological activities. In the present study, a known benzopyran derivative, (6E,10E) isopolycerasoidol (1), and a new benzopyran derivative, (6E,10E) isopolycerasoidol methyl ester (2), were isolated from a methanol extract of Pseuduvaria monticola leaves. The structures of the isolated compounds were elucidated by spectroscopic methods including 1D and 2D NMR, IR, UV, and LCMS-QTOF, and by comparison with previously published data. The anti-proliferative and cytotoxic effects of these compounds on human breast cancer cell-lines (MCF-7 and MDA-MB-231) and a human normal breast epithelial cell line (MCF-10A) were investigated. MTT results revealed both (1) and (2) were efficient in reducing cell viability of breast cancer cells. Flow cytometry analysis demonstrated that (1) and (2) induced cell death via apoptosis, as demonstrated by an increase in phosphotidylserine exposure. Both compounds elevated ROS production, leading to reduced mitochondrial membrane potential and increased plasma membrane permeability in breast cancer cells. These effects occurred concomitantly with a dose-dependent activation of caspase 3/7 and 9, a down-regulation of the anti-apoptotic gene BCL2 and the accumulation of p38 MAPK in the nucleus. Taken together, our data demonstrate that (1) and (2) induce intrinsic mitochondrial-mediated apoptosis in human breast cancer cells, which provides the first pharmacological evidence for their future development as anticancer agents.
Several chalcones were synthesized and their in vitro cytotoxicity against various human cell lines, including human breast adenocarcinoma cell line MCF-7, human lung adenocarcinoma cell line A549, human prostate cancer cell line PC3, human adenocarcinoma cell line HT-29 (colorectal cancer) and human normal liver cell line WRL-68 was evaluated. Most of the compounds being active cytotoxic agents, four of them with minimal IC₅₀ values were chosen and studied in detail with MCF-7 cells. The compounds 1, 5, 23, and 25 were capable in eliciting apoptosis in MCF-7 cells as shown by multiparameter cytotoxicity assay and caspase-3/7, -8, and -9 activities (p < 0.05). The ROS level showed 1.3-fold increase (p < 0.05) at the low concentrations used and thus it was concluded that the compounds increased the ROS level eventually leading to apoptosis in MCF-7 cells through intrinsic as well as extrinsic pathways.
Geranylated 4-phenylcoumarins, DMDP-1 & -2 isolated from Mesua elegans were investigated for anticancer potential against human prostate cancer cells. Treatment with DMDP-1 & -2 resulted in cell death in a time and dose dependent manner in an MTT assay on all cancer cell lines tested with the exception of lung adenocarcinoma cells. DMDP-1 showed highest cytotoxic efficacy in PC-3 cells while DMDP-2 was most potent in DU 145 cells. Flow cytometry indicated that both coumarins were successful to induce programmed cell death after 24 h treatment. Elucidation on the mode-of-action via protein arrays and western blotting demonstrated death induced without any significant expressions of caspases, Bcl-2 family proteins and cleaved PARP, thus suggesting the involvement of caspase-independent pathways. In identifying autophagy, analysis of GFP-LC3 showed increased punctate in PC-3 cells pre-treated with CQ and treated with DMDP-1. In these cells decreased expression of autophagosome protein, p62 and cathepsin B further confirmed autophagy. In contrary, the DU 145 cells pre-treated with CQ and treated with DMDP-2 has reduced GFP-LC3 punctate although the number of cells with obvious GFP-LC3 puncta was significantly increased in the inhibitor-treated cells. The increase level of p62 suggested leakage of cathepsin B into the cytosol to trigger potential downstream death mediators. This correlated with increased expression of cathepsin B and reduced expression after treatment with its inhibitor, CA074. Also auto-degradation of calpain-2 upon treatment with DMDP-1 &-2 and its inhibitor alone, calpeptin compared with the combination treatment, further confirmed involvement of calpain-2 in PC-3 and DU 145 cells. Treatment with DMDP-1 & -2 also showed up-regulation of total and phosphorylated p53 levels in a time dependent manner. Hence, DMDP-1 & -2 showed ability to activate multiple death pathways involving autophagy, lysosomal and endoplasmic reticulum death proteins which could potentially be manipulated to develop anti-cancer therapy in apoptosis resistant cells.
Mycobacterium indicus pranii (MIP) is a non-pathogenic mycobacterium, which has been tested on several cancer types like lung and bladder where tumour regression and complete recovery was observed. In discovering the potential cytotoxic elements, a preliminary test was carried out using four different fractions consisting of live bacteria, culture supernatant, heat killed bacteria and heat killed culture supernatant of MIP against two human cancer cells A549 and CaSki by 3-(4,5-dimethyl thiazol)-2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptosis was investigated in MCF-7 and ORL-115 cancer cells by poly-(ADP-ribose) polymerase (PARP) and DNA fragmentation assays. Among four MIP fractions, only heat killed MIP fraction (HKB) showed significant cytotoxicity in various cancer cells with inhibitory concentration, IC50 in the range 5.6-35.0 μl/(1.0 × 10(6) MIP cells/ml), while cytotoxicity effects were not observed in the remaining fractions. HKB did not show cytotoxic effects on non-cancerous cells contrary to cancerous cells, suggesting its safe usage and ability to differentially recognize between these cells. Evaluation on PARP assay further suggested that cytotoxicity in cancer cells were potentially induced via caspase-mediated apoptosis. The cytotoxic and apoptotic effects of MIP HKB have indicated that this fraction can be a good candidate to further identify effective anti-cancer agents.
Glaucoma is now regarded as a neurodegenerative disorder. A number of theories including the mechanical and vascular models have been used to explain the pathogenesis of glaucoma. However, there is now increasing evidence of biochemical molecules which may play a part in it's causation. These biochemical mechanisms include the role of excitatory aminoacids, caspases, protein kinases, oxygen free radicals, nitric oxide, TNF-alpha, neurotrophins and metalloproteins. This paper reviews these new developments which form the biochemical basis of glaucomatous neural degeneration. How to cite this article: Ahmad SS, Ghani SA, Rajagopal TH. Current Concepts in the Biochemical Mechanisms of Glaucomatous Neurodegeneration. J Current Glau Prac 2013;7(2):49-53.