Zahoor A. Wani

Plant-endophyte symbiosis, an ecological perspective

Endophytism is the phenomenon of mutualistic association of a plant with a microorganism wherein the microbe lives within the tissues of the plant without causing any symptoms of disease. In addition to being a treasured biological resource, endophytes play diverse indispensable functions in nature for plant growth, development, stress tolerance, and adaptation. Our understanding of endophytism and its ecological aspects are overtly limited, and we have only recently started to appreciate its essence. Endophytes may impact plant biology through the production of diverse chemical entities including, but not limited to, plant growth hormones and by modulating the gene expression of defense and other secondary metabolic pathways of the host. Studies have shown differential recruitment of endophytes in endophytic populations of plants growing in the same locations, indicating host specificity and that endophytes evolve in a coordinated fashion with the host plants. Endophytic technology can be employed for the efficient production of agricultural and economically important plants and plant products. The rational application of endophytes to manipulate the microbiota, intimately associated with plants, can help in enhancement of production of agricultural produce, increased production of key metabolites in medicinal and aromatic plants, as well as adaption to new bio-geographic regions through tolerance to various biotic and abiotic conditions. However, the potential of endophytic biology can be judiciously harnessed only when we obtain insight into the molecular mechanism of this unique mutualistic relationship. In this paper, we present a discussion on endophytes, endophytism, their significance, and diverse functions in nature as unraveled by the latest research to understand this universal natural phenomenon.

2-Anilinonicotinyl linked 2-aminobenzothiazoles and [1,2,4]triazolo[1,5-b] [1,2,4]benzothiadiazine conjugates as potential mitochondrial apoptotic inducers

A series of N-(2-anilino-pyridyl) linked 2-amino benzothiazoles (4a-n) and [1,2,4]triazolo [1,5-b]benzothiadiazine conjugates (5a-j) have been designed, synthesized and evaluated for their antiproliferative activity. Some of these compounds (4h-k, 4n, and 5e) have exhibited potent cytotoxicity specifically against human leukemia HL-60 cell lines with IC(50) values in the range of 0.08-0.70 μM. All these compounds were tested for their effects on the cell cycle perturbations and induction of apoptosis. Morphological evidences of apoptosis, including fragmentation of nuclei and inter nucleosomal DNA laddering formation were clearly observed after 24h exposure to compound 4i. Flow cytometry analysis revealed that compound 4i showed drastic cell cycle perturbations due to concentration dependant increase in the sub-G0 region which comprises of both the apoptotic and debris fraction, thus implying the extent of cell death. These compounds trigger the mitochondrial apoptotic pathway that results in the loss of mitochondrial membrane potential through activation of multiple caspases followed by activation of caspase-3, and finally cleavage of PARP. Further the mechanism of cell death was analysed by fluorescent microscopic analysis and also by scanning electron microscopy. The cytotoxicity of 4i correlated with induction of apoptosis, caspases activation and DNA damage and thus indicating the apoptotic pathway of anticancer effect of these compounds.

A novel parthenin analog exhibits anti-cancer activity: Activation of apoptotic signaling events through robust NO formation in human leukemia HL-60 cells

This study describes the anti-cancer activity of P19, an analog of parthenin. P19 induced apoptosis in HL-60 cells and inhibited cell proliferation with 48h IC50 of 3.5μM. At 10mg/kg dose, it doubled the median survival time of L1210 leukemic mice and at 25mg/kg it inhibited Ehrlich ascites tumor growth by 60%. Investigation of the mechanism of P19 induced apoptosis in HL-60 cells revealed that N-acetyl-l-cysteine (NAC) and s-methylisothiourea (sMIT) could reverse several molecular events that lead to cell death by inhibiting nitric oxide (NO) formation. It selectively produced massive NO in cells while quenching the basal ROS levels with concurrent elevation of GSH. P19 disrupted mitochondrial integrity leading to cytochrome c release and caspase-9 activation. P19 also caused caspase-8 activation by selectively elevating the expression of DR4 and DR5. All these events lead to the activation of caspase-3 leading to PARP-1 cleavage and DNA fragmentation. However, knocking down of AIF by siRNA also suppressed the apoptosis substantially thus indicating caspase independent apoptosis, too. Further, contrary to enhanced iNOS expression, its transcription factor, NF-κB (p65) was cleaved with a simultaneous increase in cytosolic IκB-alpha. In addition, P19 potently inhibited pro-survival proteins pSTAT3 and survivin. The multi-modal pro-apoptotic activity of P19 raises its potential usefulness as a promising anti-cancer therapeutic.

Phialomustin A–D, new antimicrobial and cytotoxic metabolites from an endophytic fungus, Phialophora mustea

Phialomustin A–D (1–4), four new bioactive metabolites, with an unprecedented azaphilone derived skeleton, were isolated and characterized from an endophytic fungus isolated from Crocus sativus. The ITS-5.8S-ITS2 ribosomal gene sequence of the endophyte displayed a sequence similarity of more than 99% with Phialophora mustea. The structural determinations of compounds (1–4) were authenticated by spectroscopic and chemical analysis. The absolute configuration of the stereogenic centers of 1, 3 and 4 were determined by electronic circular dichroism spectroscopy. Compounds 3 and 4 showed promising antifungal activities against Candida albicans, with IC50 values of 14.3 and 73.6 μM, whereas compound 2 exhibited remarkable cytotoxic activity against the human breast cancer cell line, T47D, with an IC50 of 1 μM.

The anti-angiogenic and cytotoxic effects of the boswellic acid analog BA145 are potentiated by autophagy inhibitors

BackgroundWhile angiogenesis inhibitors represent a viable cancer therapy, there is preclinical and clinical data to suggest that many tumors develop resistance to such treatments. Moreover, previous studies have revealed a complex association between autophagy and angiogenesis, and their collective influence on tumorigenesis. Autophagy has been implicated in cytoprotection and tumor promotion, and as such may represent an alternative way of targeting apoptosis-resistant cancer cells. This study explored the anti-cancer agent and boswellic acid analog BA145 as an inducer of autophagy and angiogenesis-mediated cytoprotection of tumor cells.MethodsFlow cytometry, western blotting, and confocal microscopy were used to investigate the role of BA145 mediated autophagy. ELISA, microvessel sprouting, capillary structure formation, aortic ring and wound healing assays were performed to determine the relationship between BA145 triggered autophagy and angiogenesis. Flow cytometery, western blotting, and microscopy were employed to examine the mechanism of BA145 induced cell death and apoptosis. Live imaging and tumor volume analysis were carried out to evaluate the effect of BA145 triggered autophagy on mouse tumor xenografts.ResultsBA145 induced autophagy in PC-3 cancer cells and HUVECs significantly impeded its negative regulation on cell proliferation, migration, invasion and tube formation. These effects of BA145 induced autophagy were observed under both normoxic and hypoxic conditions. However, inhibition of autophagy using either pharmacological inhibitors or RNA interference enhanced the BA145 mediated death of these cells. Similar observations were noticed with sunitinib, the anti-angiogenic properties of which were significantly enhanced during combination treatments with autophagy inhibitors. In mouse tumor xenografts, co-treatment with chloroquinone and BA145 led to a considerable reduction in tumor burden and angiogenesis compared to BA145 alone.ConclusionThese studies reveal the essential role of BA145 triggered autophagy in the regulation of angiogenesis and cytoprotection. It also suggests that the combination of the autophagy inhibitors with chemotherapy or anti-angiogenic agents may be an effective therapeutic approach against cancer.

NF-κB down-regulation and PARP cleavage by novel 3-α-butyryloxy-β-boswellic acid results in cancer cell specific apoptosis and in vivo tumor regression.

The present study relates to the induction of apoptosis thereof cytotoxicity and anti-cancer activity displayed by semi-synthetic analog of Boswellic acid i.e. 3-α-Butyryloxy-β-boswellic acid (BOBA). The cytotoxicity data revealed the differential sensitivity of cancer cell lines towards BOBA which may display its impact against different types of cancers. Considering the inhibitory potential of BOBA, we further sought to understand the target for BOBA deciphering the mechanism of action leading to apoptotic cell death and it was for the first time reported about the triterpenoid ring especially the β-boswellic acid derivative is targeting PI3K pathway. Our data revealed that BOBA treatment provides evidence about the apoptotic nature showing the potential of targeting mitochondria dependent pathways during apoptosis in HL-60 cells. BOBA induced hypo-diploid sub-G(1) DNA population in HL-60 cells as was also evident from the pattern of DNA fragmentation and mitochondrial membrane potential (ΛΨm) loss. Morphological analysis under fluorescent and scanning electron microscopy displayed typical features such as cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation. These events paralleled with the down-regulation of NF-κB and induced PARP cleavage. Furthermore, it is noteworthy that BOBA also depicted significant growth inhibition in Ehrlich Ascitic Tumour (EAT), Ehrlich Ascitic Carcinoma (EAC) and Sarcoma- 180 tumour models. Taken together, BOBA treatment may represent as potential agent to the currently available anticancer agents in both prophylactic and/or therapeutic applications. Also, our findings may open up a new perspective in the construction of novel anticancer agents based on boswellic acids that will facilitate the development of these agents for anticancer therapeutics.

In Vitro and In Vivo Anticancer Activity of Root Extracts of Sansevieria liberica Gerome and Labroy (Agavaceae)

Introduction. Sansevieria liberica Gerome and Labroy (Agavaceae) is a perennial plant widely distributed in tropical Africa. Preparations of the plant are commonly used across Nigeria for the treatment of inflammatory conditions. Based on the fact that herbal medicine is a strong component of integrative medicine, this study was conducted to evaluate the anticancer activity of root extracts of Sansevieria liberica. Methods. Sulforhodamine B (SRB) in vitro cytotoxicity assay, Sarcoma-180 (S-180) ascites and solid tumor, and L1210 lymphoid leukemia in vivo models were used in this study. Results. SL-A002 (IC50 23 µg/mL with HeLa), SL-A003 (IC50 22 µg/mL with HCT-116), and SL-A004 (IC50 23 and 18 µg/mL with A549 and THP-1, resp.) demonstrated significant activity in the SRB cytotoxicity assay. Potency was highest with the following pairs of extract : cancer cell line: SL-A002 : HeLa (IC50 23 µg/mL), SL-A003 : HCT-116 (IC50 22 µg/mL), and SL-A004 : THP-1 (IC50 18 µg/mL). SL-A002 demonstrated significant dose-dependent antitumor activity in the Sarcoma-180 (S-180) ascites model with peak effect produced at the dose of 120 mg/kg (i.p.) with inhibition of 89.36% compared to 97.96% for 5-FU (20 mg/kg i.p.). The inhibition of tumor growth by SL-A002 in the S-180 solid tumor model was 47.40% compared to a value of 50.18% for 5-FU. SL-A002 was also significantly active in the L1210 lymphoid leukemia model with 158.33% increase in mean survival time, the same value for 5-FU. Conclusions. The hydroethanolic extract of Sansevieria liberica, SL-A002, possesses significant anticancer activity to warrant further extensive study to identify, isolate, and characterize the specific bioactive molecules responsible for the observed antitumor activity and the precise mechanism(s) of action.

Anticancer activity of Aristolochia ringens Vahl. (Aristolochiaceae).

Cancer is a leading cause of death worldwide and sustained focus is on the discovery and development of newer and better tolerated anticancer drugs especially from plants. The sulforhodamine B (SRB) in vitro cytotoxicity assay, sarcoma-180 (S-180) ascites and solid tumor, and L1210 lymphoid leukemia in vivo models were used to investigate the anticancer activity of root extracts of Aristolochia ringens Vahl. (Aristolochiaceae; 馬兜鈴 mǎ dōu líng). AR-A001 (IC50 values of 20 μg/mL, 22 μg/mL, 3 μg/mL, and 24 μg/mL for A549, HCT-116, PC3, and THP-1 cell lines, respectively), and AR-A004 (IC50 values of 26 μg/mL, 19.5 μg/mL, 12 μg/mL, 28 μg/mL, 30 μg/mL, and 22 μg/mL for A549, HCT-116, PC3, A431, HeLa, and THP-1, respectively), were observed to be significantly active in vitro. Potency was highest with AR-A001 and AR-A004 for PC3 with IC50 values of 3 μg/mL and 12 μg/mL, respectively. AR-A001 and AR-A004 produced significant (p < 0.05–0.001) dose-dependent inhibition of tumor growth in the S-180 ascites model with peak effects produced at the highest dose of 120 mg/kg. Inhibition values were 79.51% and 89.98% for AR-A001 and AR-A004, respectively. In the S-180 solid tumor model, the inhibition of tumor growth was 29.45% and 50.50% for AR-A001 (120 mg/kg) and AR-A004 (110 mg/kg), respectively, compared to 50.18% for 5-fluorouracil (5-FU; 20 mg/kg). AR-A001 and AR-A004 were also significantly active in the leukemia model with 211.11% and 155.56% increase in mean survival time (MST) compared to a value of 211.11% for 5-FU. In conclusion, the ethanolic (AR-A001) and dichloromethane:methanol (AR-A004) root extracts of AR possess significant anticancer activities in vitro and in vivo.

Transcriptomic Studies Revealing Enigma of Plant-Pathogen Interaction

Plants being sessile organisms encounter numerous attacks by pathogens and pests with different lifestyles and modes of attack. In response, plants undergo cellular reprogramming in order to perceive these attacks and activate specific defense pathways. Plants possess extensive regulatory mechanisms which come into play during defense responses so as to coordinate the perception and activation of pathways specific to the type of pathogen in question. Further, many small molecule hormones play pivotal role in defense pathways and cross communicate with each other, thereby helping plant to finely regulate its response. This suggests that plant defense is controlled by intricate transcriptional regulatory network, therefore urging the need to develop genome- and transcriptome-based strategies to unravel these mechanisms. Transcriptomics has fuelled a better understanding of many biological processes and can therefore be used for understanding the host-pathogen interactions as well. Transcriptome analysis can provide more comprehensive picture of the pathways that come into play in response to different pathogens and also decipher the cascade of transcriptional events involved. This may also help in identifying the regulatory nodes in the transcriptional networks and understanding the hierarchical relationship between them. These resources in turn will help in understanding of the complex architecture of plant/host defense system which will have a long-term impact and value for crop improvement.

Porostereum sp., Associated with Saffron ( Crocus sativus L.), is a Latent Pathogen Capable of Producing Phytotoxic Chlorinated Aromatic Compounds

Saffron (Crocus sativus L.) is one of the most expensive spices in the world due to its medicinal and aromatic value. However, saffron production is severely affected by the corm rot disease throughout the saffron producing countries. In this study, we report a basidiomycetous latent pathogen of saffron, designated as CSE26, capable of producing phytotoxic compounds. CSE26 is a highly odorous basidiomycete with monomitic hyphal system. Molecular phylogeny of ITS and 28S ribosomal gene sequence of CSE26 assigned it as Porostereum spadiceum. It was found to produce corm rot in C. sativus under in vivo and field conditions, with a disease severity index of 0.7 and 0.5, respectively. CSE26 was found to produce chlorinated aromatic compounds (CAMs) having phytotoxic activity against Arabidopsis plants. Therefore, these compounds may be acting as pathogenic determinants of CSE26. However, there is a need to study the level of production of these CAMs by this fungus in the natural environment and their effects on plant health.