Evaluation of the efficacy & biochemical mechanism of cell death induction by Piper longum extract selectively in in-vitro and in-vivo models of human cancer cells.
BACKGROUND
Currently chemotherapy is limited mostly to genotoxic drugs that are associated with severe side effects due to non-selective targeting of normal tissue. Natural products play a significant role in the development of most chemotherapeutic agents, with 74.8% of all available chemotherapy being derived from natural products.


OBJECTIVE
To scientifically assess and validate the anticancer potential of an ethanolic extract of the fruit of the Long pepper (PLX), a plant of the piperaceae family that has been used in traditional medicine, especially Ayurveda and investigate the anticancer mechanism of action of PLX against cancer cells.


METHODS
Following treatment with ethanolic long pepper extract, cell viability was assessed using a water-soluble tetrazolium salt; apoptosis induction was observed following nuclear staining by Hoechst, binding of annexin V to the externalized phosphatidyl serine and phase contrast microscopy. Image-based cytometry was used to detect the effect of long pepper extract on the production of reactive oxygen species and the dissipation of the mitochondrial membrane potential following Tetramethylrhodamine or 5,5,6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine chloride staining (JC-1). Assessment of PLX in-vivo was carried out using Balb/C mice (toxicity) and CD-1 nu/nu immunocompromised mice (efficacy). HPLC analysis enabled detection of some primary compounds present within our long pepper extract.


RESULTS
Our results indicated that an ethanolic long pepper extract selectively induces caspase-independent apoptosis in cancer cells, without affecting non-cancerous cells, by targeting the mitochondria, leading to dissipation of the mitochondrial membrane potential and increase in ROS production. Release of the AIF and endonuclease G from isolated mitochondria confirms the mitochondria as a potential target of long pepper. The efficacy of PLX in in-vivo studies indicates that oral administration is able to halt the growth of colon cancer tumors in immunocompromised mice, with no associated toxicity. These results demonstrate the potentially safe and non-toxic alternative that is long pepper extract for cancer therapy.
Department of Chemistry & Biochemistry, University of Windsor, Windsor, ON, Canada.
Pamela
P
Ovadje
Department of Chemistry & Biochemistry, University of Windsor, Windsor, ON, Canada.
Dennis
D
Ma
Department of Chemistry & Biochemistry, University of Windsor, Windsor, ON, Canada.
Phillip
P
Tremblay
Department of Chemistry & Biochemistry, University of Windsor, Windsor, ON, Canada.
Alessia
A
Roma
Department of Chemistry & Biochemistry, University of Windsor, Windsor, ON, Canada.
Matthew
M
Steckle
Department of Biology, University of Ottawa, Ottawa, ON, Canada.
Jose-Antonio
JA
Guerrero
Department of Biology, University of Ottawa, Ottawa, ON, Canada.
John Thor
JT
Arnason
Department of Chemistry & Biochemistry, University of Windsor, Windsor, ON, Canada.
Siyaram
S
Pandey
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D006801:Humans; D007124:Immunoenzyme Techniques; D066298:In Vitro Techniques; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology*; D031701:Piper / Q000737:chemistry*; D010936:Plant Extracts / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
D010936:Plant Extracts; D017382:Reactive Oxygen Species