Pallab Ray

Curcumin Enhances the Efficacy of Chemotherapy by Tailoring p65NFκB-p300 Cross-talk in Favor of p53-p300 in Breast Cancer

Background: Constitutive activation of NFκB has been found in various cancers, causing resistance to chemotherapeutic drugs. Results: Curcumin pretreatment alleviates p65NFκB activation and hence tailors p65NFκB-p300 cross-talk in favor of p53-p300 in drug-resistant cells. Conclusion: This preclinical study suggests curcumin as a potent chemo-sensitizer to improve the therapeutic index. Significance: These results suggest that curcumin can be developed into an adjuvant chemotherapeutic drug. Breast cancer cells often develop multiple mechanisms of drug resistance during tumor progression, which is the major reason for the failure of breast cancer therapy. High constitutive activation of NFκB has been found in different cancers, creating an environment conducive for chemotherapeutic resistance. Here we report that doxorubicin-induced SMAR1-dependent transcriptional repression and SMAR1-independent degradation of IkBα resulted in nuclear translocation of p65NFκB and its association with p300 histone acetylase and subsequent transcription of Bcl-2 to impart protective response in drug-resistant cells. Consistently SMAR1-silenced drug-resistant cells exhibited IkBα-mediated inhibition of p65NFκB and induction of p53-dependent apoptosis. Interestingly, curcumin pretreatment of drug-resistant cells alleviated SMAR1-mediated p65NFκB activation and hence restored doxorubicin sensitivity. Under such anti-survival condition, induction of p53-p300 cross-talk enhanced the transcriptional activity of p53 and intrinsic death cascade. Importantly, promyelocyte leukemia-mediated SMAR1 sequestration that relieved the repression of apoptosis-inducing genes was indispensable for such chemo-sensitizing ability of curcumin. A simultaneous decrease in drug-induced systemic toxicity by curcumin might also have enhanced the efficacy of doxorubicin by improving the intrinsic defense machineries of the tumor-bearer. Overall, the findings of this preclinical study clearly demonstrate the effectiveness of curcumin to combat doxorubicin-resistance. We, therefore, suggest curcumin as a potent chemo-sensitizer to improve the therapeutic index of this widely used anti-cancer drug. Taken together, these results suggest that curcumin can be developed into an adjuvant chemotherapeutic drug.

Gain of Cellular Adaptation Due to Prolonged p53 Impairment Leads to Functional Switchover from p53 to p73 during DNA Damage in Acute Myeloid Leukemia Cells

Tumor suppressor p53 plays the central role in regulating apoptosis in response to genotoxic stress. From an evolutionary perspective, the activity of p53 has to be backed up by other protein(s) in case of any functional impairment of this protein, to trigger DNA damage-induced apoptosis in cancer cells. We adopted multiple experimental approaches to demonstrate that in p53-impaired cancer cells, DNA damage caused accumulation of p53 paralogue p73 via Chk-1 that strongly impacted Bax expression and p53-independent apoptosis. On the contrary, when p53 function was restored by ectopic expression, Chk-2 induced p53 accumulation that in turn overshadowed p73 activity, suggesting an antagonistic interaction between p53 family members. To understand such interaction better, p53-expressing cells were impaired differentially for p53 activity. In wild-type p53-expressing cancer cells that were silenced for p53 for several generations, p73 was activated, whereas no such trend was observed when p53 was transiently silenced. Prolonged p53 interference, even in functional p53 settings, therefore, leads to the “gain of cellular adaptation” in a way that alters the cellular microenvironment in favor of p73 activation by altering p73-regulatory proteins, e.g. Chk1 activation and dominant negative p73 down-regulation. These findings not only unveil a hitherto unexplained mechanism underlying the functional switchover from p53 to p73, but also validate p73 as a promising and potential target for cancer therapy in the absence of functional p53.

Targeting RET to induce medullary thyroid cancer cell apoptosis: an antagonistic interplay between PI3K/Akt and p38MAPK/caspase-8 pathways.

Mutations in REarranged during Transfection (RET) receptor tyrosine, followed by the oncogenic activation of RET kinase is responsible for the development of medullary thyroid carcinoma (MTC) that responds poorly to conventional chemotherapy. Targeting RET, therefore, might be useful in tailoring surveillance of MTC patients. Here we showed that theaflavins, the bioactive components of black tea, successfully induced apoptosis in human MTC cell line, TT, by inversely modulating two molecular pathways: (i) stalling PI3K/Akt/Bad pathway that resulted in mitochondrial transmembrane potential (MTP) loss, cytochrome-c release and activation of the executioner caspases-9 and -3, and (ii) upholding p38MAPK/caspase-8/caspase-3 pathway via inhibition of Ras/Raf/ERK. Over-expression of either constitutively active myristoylated-Akt-cDNA (Myr-Akt-cDNA) or dominant-negative-caspase-8-cDNA (Dn-caspase-8-cDNA) partially blocked theaflavin-induced apoptosis, while co-transfection of Myr-Akt-cDNA and Dn-caspase-8-cDNA completely eradicated the effect of theaflavins thereby negating the possibility of existence of other pathways. A search for the upstream signaling revealed that theaflavin-induced disruption of lipid raft caused interference in anchorage of RET in lipid raft that in turn stalled phosphorylation of Ras and PI3Kinase. In such anti-survival cellular micro-environment, pro-apoptotic signals were triggered to culminate into programmed death of MTC cell. These findings not only unveil a hitherto unexplained mechanism underlying theaflavin-induced MTC death, but also validate RET as a promising and potential target for MTC therapy.

ROS-PIASγ cross talk channelizes ATM signaling from resistance to apoptosis during chemosensitization of resistant tumors.

With the existing knowledge of ATM’s role in therapeutic resistance, the present study aimed at identifying the molecular mechanisms that influence ATM to oscillate between chemoresistance and chemosensitivity. We observed that the redox status of tumors functions as a major determinant of ATM-dependent ‘resistance-to-apoptosis’ molecular switch. At a low reactive oxygen species (ROS) condition during genotoxic insult, the ATM/sumoylated-IKKγ interaction induced NFκB activation that resisted JNK-mediated apoptosis, whereas increasing cellular ROS restored ATM/JNK apoptotic signaling. A search for the upstream missing link revealed that high ROS induces oxidation and ubiquitin-mediated degradation of PIASγ, thereby disrupting PIASγ-IKKγ cross talk, a pre-requisite for IKKγ sumoylation and subsequent NFκB activation. Interruption in the PIASγ-mediated resistance pathway channels ATM signaling toward ATM/JNK pro-death circuitry. These in vitro results also translated to sensitive and resistant tumor allograft mouse models in which low ROS-induced resistance was over-ruled in PIASγ knockout tumors, while its overexpression inhibited high ROS-dependent apoptotic cues. Cumulatively, our findings identified an unappreciated yet critical combinatorial function of cellular ROS and PIASγ in regulating ATM-mediated chemosensitization of resistant tumors. Thus, therapeutic strategies employing ROS upregulation to inhibit PIASγ during genotoxic therapy may, in future, help to eliminate the problems of NFκB-mediated tumor drug resistance.

Crocetin exploits p53-induced death domain (PIDD) and FAS-associated death domain (FADD) proteins to induce apoptosis in colorectal cancer

Tumor suppressor p53 preserves the genomic integrity by restricting anomaly at the gene level. The hotspots for mutation in half of all colon cancers reside in p53. Hence, in a p53-mutated cellular milieu targeting cancer cells may be achievable by targeting the paralogue(s) of p53. Here we have shown the effectiveness of crocetin, a dietary component, in inducing apoptosis of colon cancer cells with varying p53 status. In wild-type p53-expressing cancer cells, p53 in one hand transactivates BAX and in parallel up-regulates p53-induced death domain protein (PIDD) that in turn cleaves and activates BID through caspase-2. Both BAX and t-BID converge at mitochondria to alter the transmembrane potential thereby leading to caspase-9 and caspase-3-mediated apoptosis. In contrast, in functional p53-impaired cells, this phytochemical exploits p53-paralogue p73, which up-regulates FAS to cleave BID through FAS-FADD-caspase-8-pathway. These findings not only underline the phenomenon of functional switch-over from p53 to p73 in p53-impaired condition, but also validate p73 as a promising and potential target for cancer therapy in absence of functional p53.

Multilocus sequence analysis reveals high genetic diversity in clinical isolates of Burkholderia cepacia complex from India

Burkholderia cepacia complex (Bcc) is a complex group of bacteria causing opportunistic infections in immunocompromised and cystic fibrosis (CF) patients. Herein, we report multilocus sequence typing and analysis of the 57 clinical isolates of Bcc collected over the period of seven years (2005–2012) from several hospitals across India. A total of 21 sequence types (ST) including two STs from cystic fibrosis patient’s isolates and twelve novel STs were identified in the population reflecting the extent of genetic diversity. Multilocus sequence analysis revealed two lineages in population, a major lineage belonging to B. cenocepacia and a minor lineage belonging to B. cepacia. Split-decomposition analysis suggests absence of interspecies recombination and intraspecies recombination contributed in generating genotypic diversity amongst isolates. Further linkage disequilibrium analysis indicates that recombination takes place at a low frequency, which is not sufficient to break down the clonal relationship. This knowledge of the genetic structure of Bcc population from a rapidly developing country will be invaluable in the epidemiology, surveillance and understanding global diversity of this group of a pathogen.

Changing epidemiology of coagulase-negative staphylococci in normal flora of skin

Coagulase negative staphylococci (CoNS) have recently emerged as important pathogens causing nosocomial blood stream infections. To evaluate the prevalence of CoNS in cutaneous normal flora, skin swabs were collected from voluntary blood donors and processed for culture and identification using matrix assisted laser desorption ionisation-time of flight (MALDI-TOF). CoNS were isolated from 96% of blood donors, most commonly Staphylococcus hominis (86%), followed by Staphylococcus epidermidis (22%) and Staphylococcus haemolyticus (9%). There has been a shift in the prevalent species of CoNS in the community in India, from the earlier known S. epidermidis towards resistant species such as S. hominis and S. haemolyticus. Routine and rapid speciation of CoNS in clinical isolates with MALDI-TOF should be used effectively to manage these resistant species.

MALDI-TOF mass spectrometry: An emerging tool for unequivocal identification of non-fermenting Gram-negative bacilli

Background & objectives: Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been instrumental in revolutionizing microbiological identification, especially in high-throughput laboratories. It has enabled the identification of organisms like non-fermenting Gram-negative bacilli (NFGNB), which has been a challenging task using conventional methods alone. In this study an attempt was made to validate MALDI-TOF MS for the identification of clinical isolates of each of the three most common NFGNB, other than Pseudomonas spp., taking molecular methods as the gold standard. Methods: One hundred and fifty clinical isolates of NFGNB, confirmed by molecular methods such as Acinetobacter baumannii[oxa-51 polymerase chain reaction (PCR)], Burkholderia cepacia complex (expanded multilocus sequence typing) and Stenotrophomonas maltophilia (species-specific PCR), were taken. Isolated colonies from fresh cultures of all 150 isolates were smeared onto ground steel plate, with and without formic acid extraction step. The identification was carried out using MALDI-TOF MS Biotyper database. Results: A concordance of 100 and 73.33 per cent was found between the molecular techniques and MALDI-TOF MS system in the identification of these isolates up to genus and species levels, respectively. Using a cut-off of 1.9 for reliable identification, rate of species identification rose to 82.66 per cent. Principal component analysis dendrogram and cluster analysis further increased discrimination of isolates. Interpretation & conclusions: Our findings showed MALDI-TOF MS-based identification of NFGNB as a good, robust method for high-throughput laboratories.

Naso-pharyngeal carriage of organisms in children aged 3–59 months diagnosed with severe community-acquired pneumonia

ObjectiveTo study the naso-pharyngeal carriage of organisms in children diagnosed with severe pneumonia.MethodsNasopharyngeal aspirate and swabs for microbiological analyses were collected from 377 children aged 3–59 months with severe pneumonia.Results28.6% of the samples were positive for S. pneumoniae, 9.6% were positive for H. influenzae, and 8.5% were positive for both the organisms. Respiratory syncytial virus was detected in 27% of samples. The rate of isolation of S. pneumonia and H. influenzae was significantly more in the age group of 12–59 months.ConclusionsIn children with severe pneumonia, most common organisms isolated/detected from naso-pharyngeal aspirates were S.pneumoniae and Respiratory Syncytial Virus.

Emerging metronidazole resistance in Bacteroides spp. and its association with nim gene: a study from North India

OBJECTIVES Bacteroides spp. are anaerobic resident intestinal flora but are also known to cause severe morbidity. They are resistant to a wide variety of antimicrobial agents, including metronidazole, which has been shown to be associated with specific nitroimidazole (nim) resistance genes. Metronidazole resistance is emerging worldwide, although presently it remains at ca. 5%. This study aimed to determine the metronidazole susceptibility and distribution of nim genes in Bacteroides spp. clinical isolates in India. The relationship among strains harbouring nim genes and their susceptibility to metronidazole was also analysed. METHODS A total of 42 Bacteroides spp. clinical isolates were identified using an advanced MALDI-TOF system. Minimum inhibitory concentrations (MICs) for metronidazole were determined by the agar dilution method. Bacterial DNA was extracted and was subjected to nim gene PCR and the amplified PCR products were sequenced to determine the prevalent nim types. RESULTS Bacteroides fragilis was the most common isolate (64%) among all Bacteroides spp. isolates. Among the total 42 clinical Bacteroides spp. isolates, 29 (69%) were susceptible and 13 (31%) were resistant to metronidazole by the agar dilution method. nim gene PCR performed on 38 isolates showed positivity in 20 isolates (53%), of which 12 had high metronidazole MICs (χ2 test, P<0.005). On sequencing, these nim genes were most closely related to nimE type. CONCLUSION Resistance to metronidazole is consistently emerging worldwide. There is a significant association of the nim gene with metronidazole resistance. Periodic surveillance is needed to detect geographic and temporal trends in nim gene prevalence.