Glucocorticoid receptor mediated sensitization of colon cancer to photodynamic therapy induced cell death.

Photodynamic therapy (PDT) is a clinically approved, non-invasive alternate cancer therapy. A synthetic glucocorticoid (GC), dexamethasone (Dex) has previously been demonstrated to sensitize cancer cells to chemotherapy. However, to the best of our knowledge, the sensitization effect of GCs on PDT has not yet been investigated. We hypothesized that glucocorticoid receptor (GR) targeting can selectively make cancer cells more sensitive to PDT treatment, as PDT induces hypoxia wherein GR-activity gets enhanced. In addition, Dex was reported to act against the PDT-induced cell survival pathways like HIF-1α, NRF2, NF-κB, STAT3 etc. Thus, both the treatments can complement each other and may result in increasing the effectiveness of combination therapy. Hence, in this study, we developed liposomal formulations of our previously reported PDT agent P-Nap, either alone (D1P-Nap) or in combination with Dex (D1XP-Nap) to elucidate the sensitization effect. Interestingly, our RT-PCR results in hypoxic conditions showed down-regulation of HIF-1α and over expression of GR-activated genes for glucose-6-phosphatase (G6Pase) and PEPCK enzymes, indicating prominent GR-transactivation. We also observed higher phototoxicity in CT26.WT cells treated with D1XP-Nap PDT under hypoxic conditions as compared to normoxic conditions. These effects were reversed when cells were pre-treated with RU486, a competitive inhibitor of GCs. Moreover, our in vivo findings of subcutaneous tumor model of Balb/C mice for colon cancer revealed a significant decrease in tumor volume as well as considerable enhancement in the survivability of PDT treated tumor-bearing mice when Dex was present in the formulation. A high Bax/Bcl-xL ratio, high p53 expression, enhanced E-cadherin expression and down-regulation of pro-tumorigenic transcription factors NF-κB and c-Myc were found in tumor lysates from mice treated with D1XP-Nap under PDT, indicating GR-mediated sensitization of the tumor to PDT-induced cell death and enhancement of life-span for tumor bearing mice.

Various Extraction Techniques of Curcumin-A Comprehensive Review.

Curcumin, the active component of the rhizome of Curcuma longa, is a safe substance whose applications are extensively used in medicinal, biological, pharmacological activities, and food cosmetic additives. In the field of medicine, curcuminoids have a greater impact; they have been associated with the suppression of neuropathic pain, depression, angiogenesis, tumorigenesis, diabetes, and diseases of the liver, skin, and pulmonary systems, as well as cardiovascular and nervous systems. These are in high demand and have high market potential and inflated costs. For the aforementioned uses, as well as for basic research, it is crucial to get pure curcumin from plant sources. There is a need for effective extraction and purification techniques that adhere to standards for process efficiency, environmental friendliness, and safety. Scope: This account offers an accurate and thorough explanation of the many techniques used to extract and purify curcumin from plant sources, as well as a look at its various roles in the pharmaceutical, cosmetic, medical, and other industries. Curcumin's prospective and commercial roles are also discussed. Key findings: Curcuminoids have been extracted and purified by using a broad range of techniques that are utilized extensively across the world. Extraction of curcuminoids includes both traditional and contemporary approaches, of which a handful include Soxhlet extraction, maceration, solvent extraction, ultrasound-assisted extraction, microwave-assisted extraction, enzyme-assisted extraction, and supercritical liquid extraction. The other process called purification can be performed alone or in combination with techniques. The use of column chromatography and semipreparative high-performance liquid chromatography are examples of traditional purification procedures, and other innovative methods include high-speed counter-current chromatography and supercritical fluid chromatography.

Potentiation of novel porphyrin based photodynamic therapy against colon cancer with low dose doxorubicin and elucidating the molecular signalling pathways responsible for relapse.

Photodynamic therapy (PDT) is a promising non-invasive treatment modality for cancer and can be potentiated by combination with chemotherapy. Here, we combined PDT of novel porphyrin-based photosensitizers with low dose doxorubicin (Dox) to get maximum outcome. Dox potentiated and showed synergism with PDT under in vitro conditions on CT26.WT cells. The current colon cancer treatment strategies assure partial or even complete tumour regression but loco-regional relapse or distant metastasis is the major cause of death despite combination therapy. The spared cells after the treatment contribute to relapse and it is important to study their behaviour in host environment. Hence, we developed relapse models for PDT, Dox and combination treatments by transplanting respectively treated equal number of live cells to mice (n = 5) for tumour formation. Most of the treated cells lost tumour forming ability, but some treatment resistant cells developed tumours in few mice. These tumours served as relapse models and Western blot analysis of tumour samples provided clinically relevant information to delineate resistance strategies of individual as well as combination therapies at molecular level. Our results showed that low dose Dox helped in increasing the tumour inhibiting effect of PDT in combination therapy, but still there are indeed possibilities of relapse at later stages due to chemoresistance and immune suppression that may occur post-treatment. We observed that the combination therapy may also lead to the development of multidrug resistant (MDR) phenotype during relapse. Thus, this study provided clinically relevant information to further strengthen and improve PDT-drug combination therapy in order to avoid relapse and to treat cancer more effectively.

Benzimidazole-1,2,3-triazole hybrid molecules: synthesis and study of their interaction with G-quadruplex DNA.

A series of new benzimidazole-1,2,3-triazole hybrid derivatives have been synthesized via 'click' reaction and evaluated for their in vitro cytotoxicity as well as DNA binding affinity. MTT assay showed that all the six compounds are cytotoxic to PC3 and B16-F10 cancer cell lines. Though all the compounds showed moderate interaction with G4, c-Myc promoter DNA and dsDNA, 4f exhibited selective interaction with G-quadruplex DNA over duplex DNA as demonstrated by spectroscopic experiments like UV-vis spectroscopy, fluorescence spectroscopy, CD spectroscopy, thermal melting and fluorescence lifetime experiments. They also confirm the G-quadruplex DNA stabilizing potential of 4f. Viscosity measurements also confirm that 4f exhibits high G-quadruplex DNA selectivity over duplex DNA. Docking studies supported the spectroscopic observations. Cell cycle analysis showed that 4f induces G2/M phase arrest and induces apoptosis. Hence, from these experimental results it is evident that compound 4f may be a G-quadruplex DNA groove binding molecule with anticancer activity.

Novel Amphiphilic G-Quadruplex Binding Synthetic Derivative of TMPyP4 and Its Effect on Cancer Cell Proliferation and Apoptosis Induction.

Porphyrins are well-known anticancer agents because of their high binding affinity for G-quadruplex DNA and excellent photophysical properties. Several studies carried out using TMPyP4 established it as an efficient chemotherapeutic and a photodynamic therapeutic (PDT) agent, but its use as a lead molecule has been restricted because of its high level of binding to double-stranded DNA (dsDNA), which may have side effects on normal cells and tissues. To minimize its interaction with dsDNA and to enhance internalization into cells, an analogue of TMPyP4 (5Me) was synthesized. Its selectivity for G-quadruplex DNA over dsDNA was evaluated by spectroscopic methods, and its role in stabilizing G-quadruplex DNA was assessed by fluorescence lifetime and thermal melting experiments. Biophysical studies indicated that 5Me interacts well with G-quadruplex DNA. In vitro cytotoxicity experiments with tumor cell lines (PANC-1, B16F10, and MDA MB 231) have revealed that 5Me can inhibit the growth of cancer cells comparable to TMPyP4. MTT and apoptotic assays demonstrated the ability of 5Me to specifically affect cancer cells over normal cells. Cell cycle analysis showed that 5Me, like TMPyP4, induces G2/M phase cell cycle arrest. In addition, 5Me is more effectively taken up by both cancer and normal cells than TMPyP4. In addition, we have noticed that 5Me is more efficient than TMPyP4 in inhibiting the growth of the cancer cells after irradiation with light (600-720 nm, 20 J/cm2, 50 mW/cm2). By and large, these experimental results indicate that 5Me can be an efficient chemotherapeutic as well as a PDT agent.

Photodynamic Therapy: Past, Present and Future.

Though we crossed many milestones in the field of medicine and health care in eradicating some deadly diseases over the past decades, cancer remained a challenge taking the lives of millions of people and having adverse effects on the quality of life of survivors. Chemotherapy and radiotherapy, the two existing major treatment modalities, have severe side effects and patients undergoing these treatments experience unbearable pain. Consequently, clinicians and researchers are working for the alternate treatment regimens, which can provide complete cure with minimum or no side effects. To this end, the present review highlights the major advances and future promises of photodynamic therapy, an emerging and promising therapeutic modality for combating cancer. We delve on various important aspects of photodynamic therapy including principle, mechanism of action, brief history and development of photosensitizers from first generation to the existing third generation, delivery strategies, development or suppression of immunity, combination therapy and future prospects.

Synthesis and functional characterization of a fluorescent peptide probe for non invasive imaging of collagen in live tissues.

Targeted molecular imaging to detect changes in the structural and functional organization of tissues, at the molecular level, is a promising approach for effective and early diagnosis of diseases. Quantitative and qualitative changes in type I collagen, which is a major component in the extra cellular matrix (ECM) of skin and other vital organs like lung, liver, heart and kidneys, are often associated with the pathophysiology of these organs. We have synthesized a fluorescent probe that comprises collagelin, a specific collagen binding peptide, coupled to fluorescent porphyrin that can effectively detect abnormal deposition of collagen in live tissues by emitting fluorescence in the near infra red (NIR) region. In this report we have presented the methodology for coupling of 5-(4-carboxy phenyl)-10, 15, 20-triphenyl porphyrin (C-TPP) to the N-terminal of collagelin or to another mutant peptide (used as a control). We have evaluated the efficacy of these fluorescent peptides to detect collagen deposition in live normal and abnormal tissues. Our results strongly suggest that porphyrin-tagged collagelin can be used as an effective probe for the non invasive in vivo detection of tissue fibrosis, especially in the liver.

Unfolding of in planta activity of anti-rep ribozyme in presence of a RNA silencing suppressor.

Antisense RNA ribozymes have intrinsic endonucleolytic activity to effect cleavage of the target RNA. However, this activity in vivo is often controlled by the dominance of antisense or other double-stranded RNA mechanism. In this work, we demonstrate the in planta activity of a hammerhead ribozyme designed to target rep-mRNA of a phytopathogen Mungbean Yellow Mosaic India virus (MYMIV) as an antiviral agent. We also found RNA-silencing is induced on introduction of catalytically active as well as inactive ribozymes. Using RNA-silencing suppressors (RSS), we demonstrate that the endonucleolytic activity of ribozymes is a true phenomenon, even while a mutated version may demonstrate a similar down-regulation of the target RNA. This helps to ease the confusion over the action mechanism of ribozymes in vivo.

Intervention of geminiviral replication in yeast by ribozyme mediated downregulation of its Rep protein.

Geminiviruses pose serious threat to many economically important crops such as mungbean, tomato, cotton, etc. To devise a specific antiviral strategy at the viral DNA replication level, a hammerhead ribozyme was directed against the mRNA of the replication initiator protein (Rep). Rep is the most important viral protein for the DNA replication of the Mungbean yellow mosaic India virus (MYMIV), a member of the Geminiviridae family. The ribozyme showed approximately 33% cleavage activity on synthetic rep transcript within 1h under in vitro conditions, whereas the mutant ribozyme, designed to lack the catalytic activity but target the same site, showed no cleavage. The in vivo efficiency of ribozyme was evaluated in Saccharomyces cerevisiae as it can act as a surrogate host for replication of the MYMIV-DNA and lacks RNAi machinery. In the presence of the ribozyme, growth of the yeast cells that are dependent on geminiviral replication was inhibited by 30% and cellular generation time was increased by 2h. The RT-PCR analysis showed a maximum of about 50% reduction in the rep mRNA level in presence of the ribozyme compared to its noncatalytic mutant control. About 65% decrease in geminiviral DNA replication was observed due to the downregulation of replication initiator protein by the ribozyme. These results raise the possibility of engineering resistance to geminiviruses employing the ribozyme approach.