Calotropis Procera Induced Caspase-Dependent Apoptosis and Impaired Akt/mTOR Signaling in 4T1 Breast Cancer Cells.

INTRODUCTION: Calotropis procera (Aiton) Dryand (Apocynaceae) is an herb that has been commonly used in folk medicine to treat various diseases for more than 1500 years. AIMS: Our goal was to investigate the anti-metastatic effects of phenolics extracted from C. procera (CphE) against 4T1 breast cancer cells and in BALB/c mice. METHODS: 4T1 cells were treated with CphE and quercetin (positive control) at concentrations that inhibited cell viability by 50% (IC50). Levels of reactive oxygen species (ROS), wound healing, and protein expressions were determined following standard protocols. For the in vivo pilot study, the syngeneic BALB/c mouse model was used. 4T1 cells were injected into mammary fat pads. Tumors were allowed to grow for 9 days before gavage treatment with CphE (150 mg GAE/kg/day) or PBS (controls) for one week. Excised tumors, liver, and lungs were analyzed for gene and protein expression and histology. RESULTS: In vitro results showed that CphE suppressed cell viability through apoptosis induction, via caspase-3 cleavage and total PARP reduction. CphE also scavenged ROS and suppressed Akt, mTOR, ERK1/2, CREB, and Src activation contributing to cell motility inhibition. CphE reduced IR, PTEN, TSC2, p70S6, and RPS6, protein levels, which are proteins involved in the PI3K/Akt/mTOR pathway, suggesting this pathway as CphE primary target. In vivo results showed downregulation of ERK1/2 activation by phosphorylation in tumor tissues, accompanied by angiogenesis reduction in tumor and lung tissues. A reduction of Cenpf mRNA levels in liver and lung tissues strongly suggested anti-invasive cancer activity of CphE. CONCLUSION: CphE inhibited 4T1 cell signal pathways that play a key role in cell growth and invasion. The potential for in vitro results to be translated in vivo was confirmed. A complete animal study is a guarantee to confirm the CphE anticancer and antimetastatic activity in vivo.

Population Genetics of Calotropis gigantea, a Medicinal and Fiber Resource Plant, as Inferred from Microsatellite Marker Variation in two Native Countries.

Calotropis gigantea is well known for its aesthetic, medicinal, pharmacological, fodder, fuel, and fiber production potential. Unfortunately, this plant species is still undomesticated, and the genetic information available for crop improvement is limited. For this study, we sampled 21 natural populations of C. gigantea from two key areas of its natural distribution range (Bangladesh and China) and genotyped 379 individuals using nine nuclear microsatellite markers. Population genetic diversity was higher in Bangladesh than that observed in Chinese populations. Overall, a moderate level of genetic diversity was found (Na = 3.73, HE = 0.466), with most of the genetic variation detected within populations (65.49%) and substantial genetic differentiation (FST = 0.345) between the study regions. We observed a significant correlation between genetic and geographic distances (r = 0.287, P = 0.001). The Bayesian clustering, UPGMA tree, and PCoA analyses yielded three distinct genetic pools, but the number of migrants per generation was high (NM = 0.52-2.78) among them. Our analyses also revealed that some populations may have experienced recent demographic bottlenecks. Our study provides a baseline for exploitation of the genetic resources of C. gigantea in domestication and breeding programs as well as some insights into the germplasm conservation of this valuable plant.

Genome Assembly and Annotation of the Medicinal Plant Calotropis gigantea, a Producer of Anticancer and Antimalarial Cardenolides.

Calotropis gigantea produces specialized secondary metabolites known as cardenolides, which have anticancer and antimalarial properties. Although transcriptomic studies have been conducted in other cardenolide-producing species, no nuclear genome assembly for an Asterid cardenolide-producing species has been reported to date. A high-quality de novo assembly was generated for C. gigantea, representing 157,284,427 bp with an N50 scaffold size of 805,959 bp, for which quality assessments indicated a near complete representation of the genic space. Transcriptome data in the form of RNA-sequencing libraries from a developmental tissue series was generated to aid the annotation and construction of a gene expression atlas. Using an ab initio and evidence-driven gene annotation pipeline, 18,197 high-confidence genes were annotated. Homologous and syntenic relationships between C. gigantea and other species within the Apocynaceae family confirmed previously identified evolutionary relationships, and suggest the emergence or loss of the specialized cardenolide metabolites after the divergence of the Apocynaceae subfamilies. The C. gigantea genome assembly, annotation, and RNA-sequencing data provide a novel resource to study the cardenolide biosynthesis pathway, especially for understanding the evolutionary origin of cardenolides and the engineering of cardenolide production in heterologous organisms for existing and novel pharmaceutical applications.

Transcriptome and Metabolite analysis reveal candidate genes of the cardiac glycoside biosynthetic pathway from Calotropis procera.

Calotropis procera is a medicinal plant of immense importance due to its pharmaceutical active components, especially cardiac glycosides (CG). As genomic resources for this plant are limited, the genes involved in CG biosynthetic pathway remain largely unknown till date. Our study on stage and tissue specific metabolite accumulation showed that CG's were maximally accumulated in stems of 3 month old seedlings. De novo transcriptome sequencing of same was done using high throughput Illumina HiSeq platform generating 44074 unigenes with average mean length of 1785 base pair. Around 66.6% of unigenes were annotated by using various public databases and 5324 unigenes showed significant match in the KEGG database involved in 133 different pathways of plant metabolism. Further KEGG analysis resulted in identification of 336 unigenes involved in cardenolide biosynthesis. Tissue specific expression analysis of 30 putative transcripts involved in terpenoid, steroid and cardenolide pathways showed a positive correlation between metabolite and transcript accumulation. Wound stress elevated CG levels as well the levels of the putative transcripts involved in its biosynthetic pathways. This result further validated the involvement of identified transcripts in CGs biosynthesis. The identified transcripts will lay a substantial foundation for further research on metabolic engineering and regulation of cardiac glycosides biosynthesis pathway genes.

Cysteine Protease Profiles of the Medicinal Plant Calotropis procera R. Br. revealed by de novo transcriptome analysis.

Calotropis procera R. Br., a traditional medicinal plant in India, is a promising source of commercial proteases, because the cysteine proteases from the plant exhibit high thermo-stability, broad pH optima, and plasma-clotting activity. Though several proteases such as Procerain, Procerain B, CpCp-1, CpCp-2, and CpCp-3 have been isolated and characterized, the information of their transcripts is limited to cDNAs encoding their mature peptides. Due to this limitation, in this study, to determine the cDNA sequences encoding full open reading frame of these cysteine proteases, transcripts were sequenced with an Illumina Hiseq2000 sequencer. A total of 171,253,393 clean reads were assembled into 106,093 contigs with an average length of 1,614 bp and an N50 of 2,703 bp, and 70,797 contigs with an average length of 1,565 bp and N50 of 2,082 bp using Trinity and Velvet-Oases software, respectively. Among these contigs, we found 20 unigenes related to papain-like cysteine proteases by BLASTX analysis against a non-redundant NCBI protein database. Our expression analysis revealed that the cysteine protease contains an N-terminal pro-peptide domain (inhibitor region), which is necessary for correct folding and proteolytic activity. It was evident that expression yields using an inducible T7 expression system in Escherichia coli were considerably higher with the pro-peptide domain than without the domain, which could contribute to molecular cloning of the Calotropis procera protease as an active form with correct folding.

Characterization of P5CS gene in Calotropis procera plant from the de novo assembled transcriptome contigs of the high-throughput sequencing dataset.

The wild plant known as Calotropis procera is important in medicine, industry and ornamental fields. Due to spread in areas that suffer from environmental stress, it has a large number of tolerance genes to environmental stress such as drought and salinity. Proline is one of the most compatible solutes that accumulate widely in plants to tolerate unfavorable environmental conditions. Plant proline synthesis depends on Δ-pyrroline-5-carboxylate synthase (P5CS) gene. But information about this gene in C. procera is unavailable. In this study, we uncovered and characterized P5CS (P5CS, NCBI accession no. KJ020750) gene in this medicinal plant from the de novo assembled transcriptome contigs of the high-throughput sequencing dataset. A number of GenBank accessions for P5CS sequences were blasted with the recovered de novo assembled contigs. Homology modeling of the deduced amino acids (NCBI accession No. AHM25913) was further carried out using Swiss-Model, accessible via the EXPASY. Superimposition of C. procera P5CS-like full sequence model on Homo sapiens (P5CS_HUMAN, UniProt protein accession no. P54886) was constructed using RasMol and Deep-View programs. The functional domains of the novel P5CS amino acids sequence were identified from the NCBI conserved domain database (CDD) that provide insights into sequence structure/function relationships, as well as domain models imported from a number of external source databases (Pfam, SMART, COG, PRK, TIGRFAM).

Detection of a Usp-like gene in Calotropis procera plant from the de novo assembled genome contigs of the high-throughput sequencing dataset.

The wild plant species Calotropis procera (C. procera) has many potential applications and beneficial uses in medicine, industry and ornamental field. It also represents an excellent source of genes for drought and salt tolerance. Genes encoding proteins that contain the conserved universal stress protein (USP) domain are known to provide organisms like bacteria, archaea, fungi, protozoa and plants with the ability to respond to a plethora of environmental stresses. However, information on the possible occurrence of Usp in C. procera is not available. In this study, we uncovered and characterized a one-class A Usp-like (UspA-like, NCBI accession No. KC954274) gene in this medicinal plant from the de novo assembled genome contigs of the high-throughput sequencing dataset. A number of GenBank accessions for Usp sequences were blasted with the recovered de novo assembled contigs. Homology modelling of the deduced amino acids (NCBI accession No. AGT02387) was further carried out using Swiss-Model, accessible via the EXPASY. Superimposition of C. procera USPA-like full sequence model on Thermus thermophilus USP UniProt protein (PDB accession No. Q5SJV7) was constructed using RasMol and Deep-View programs. The functional domains of the novel USPA-like amino acids sequence were identified from the NCBI conserved domain database (CDD) that provide insights into sequence structure/function relationships, as well as domain models imported from a number of external source databases (Pfam, SMART, COG, PRK, TIGRFAM).