Programmed Cell Death (PCD) in Plant: Molecular Mechanism, Regulation, and Cellular Dysfunction in Response to Development and Stress

Programmed cell death (PCD) or apoptosis is a genetically programmed cellular process. Though in the plant, a true caspase system is lacking, still PCD can occur throughout the life cycle at any cell type, tissue, and organ part in response to a wide range of stimuli. Here we have discussed the current understanding of plant PCD in terms of different pathways, cellular dysfunction, regulation, and signaling mechanisms. Our present study discussed how and to what extent PCD is involved in pre-zygotic and post-zygotic plant life cycle and emphasized to what extent PCD modulated in response to abiotic and biotic stress. Additionally, the expression profile of different PCD-associated genes that are modulated by developmental stage, biotic-abiotic stress, cellular metabolites are also elucidated. Hence, this study will be helpful for understanding the molecular and structural instincts of PCD in different stages of plant growth and development, response to biotic/abiotic stimuli, and cellular dysfunction

Tumor-Shed PGE2 Impairs IL2Rγc-Signaling to Inhibit CD4+ T Cell Survival: Regulation by Theaflavins

BACKGROUND:Many tumors are associated with decreased cellular immunity and elevated levels of prostaglandin E2 (PGE2), a known inhibitor of CD4+ T cell activation and inducer of type-2 cytokine bias. However, the role of this immunomodulator in the survival of T helper cells remained unclear. Since CD4+ T cells play critical roles in cell-mediated immunity, detail knowledge of the effect tumor-derived PGE2 might have on CD4+ T cell survival and the underlying mechanism may, therefore, help to overcome the overall immune deviation in cancer. METHODOLOGY/PRINCIPAL FINDINGS:By culturing purified human peripheral CD4+ T cells or Jurkat cells with spent media of theaflavin- or celecoxib-pre-treated MCF-7 cells, we show that tumor-shed PGE2 severely impairs interleukin 2 receptor gammac (IL2Rgammac)-mediated survival signaling in CD4+ T cells. Indeed, tumor-shed PGE2 down-regulates IL2Rgammac expression, reduces phosphorylation as well as activation of Janus kinase 3 (Jak-3)/signal transducer and activator of transcription 5 (Stat-5) and decreases Bcl-2/Bax ratio thereby leading to activation of intrinsic apoptotic pathway. Constitutively active Stat-5A (Stat-5A1 6) over-expression efficiently elevates Bcl-2 levels in CD4+ T cells and protects them from tumor-induced death while dominant-negative Stat-5A over-expression fails to do so, indicating the importance of Stat-5A-signaling in CD4+ T cell survival. Further support towards the involvement of PGE2 comes from the results that (a) purified synthetic PGE2 induces CD4+ T cell apoptosis, and (b) when knocked out by small interfering RNA, cyclooxygenase-2 (Cox-2)-defective tumor cells fail to initiate death. Interestingly, the entire phenomena could be reverted back by theaflavins that restore cytokine-dependent IL2Rgammac/Jak-3/Stat-5A signaling in CD4+ T cells thereby protecting them from tumor-shed PGE2-induced apoptosis. CONCLUSIONS/SIGNIFICANCE:These data strongly suggest that tumor-shed PGE2 is an important factor leading to CD4+ T cell apoptosis during cancer and raise the possibility that theaflavins may have the potential as an effective immunorestorer in cancer-bearer

Intra-tumor ROS amplification by melatonin interferes in the apoptosis-autophagy-inflammation-EMT collusion in the breast tumor microenvironment

Epidemiological as well as experimental studies have established that the pineal hormone melatonin has inhibitory effects on different types of cancers. Several mechanisms have been proposed for the anticancer activities of melatonin, but the fundamental molecular pathways still require clarity. We developed a mouse model of breast cancer using Ehrlich's ascites carcinoma (injected in the 4th mammary fat pad of female Swiss albino mice) and investigated the possibility of targeting the autophagy-inflammation-EMT colloquy to restrict breast tumor progression using melatonin as intervention. Contrary to its conventional antioxidant role, melatonin was shown to augment intracellular ROS and initiate ROS-dependent apoptosis in our system, by modulating the p53/JNK & NF-κB/pJNK expressions/interactions. Melatonin-induced ROS promoted SIRT1 activity. Interplay between SIRT1 and NF-κB/p65 is known to play a pivotal role in regulating the crosstalk between autophagy and inflammation. Persistent inflammation in the tumor microenvironment and subsequent activation of the IL-6/STAT3/NF-κB feedback loop promoted EMT and suppression of autophagy through activation of PI3K/Akt/mTOR signaling pathway. Melatonin disrupted NF-κB/SIRT1 interactions blocking IL-6/STAT3/NF-κB pathway. This led to reversal of pro-inflammatory bias in the breast tumor microenvironment and augmented autophagic responses. The interactions between p62/Twist1, NF-κB/Beclin1 and NF-κB/Slug were altered by melatonin to strike a balance between autophagy, inflammation and EMT, leading to tumor regression. This study provides critical insights into how melatonin could be utilized in treating breast cancer via inhibition of the PI3K/Akt/mTOR signaling and differential modulation of SIRT1 and NF-κB proteins, leading to the establishment of apoptotic and autophagic fates in breast cancer cells

Radiosensitizing effect of ellagic acid on growth of Hepatocellular carcinoma cells: an in vitro study

Abstract Failure of treatment for cancer in clinic by radio/chemotherapy is generally attributed to tumour resistance. Therefore, it is important to develop strategies to increase the cytotoxicity of tumour cells by radiation in combination with unique tumour selective cytotoxic agents. We evaluated the potential of ellagic acid (EA) as an enhancer of oxidative stress in cancer cells. HepG2 cells were treated with EA (10 µM) for 12 h prior to exposure of single 7.5 Gy dose of irradiation. Treatment of HepG2 cells with EA and gamma radiation showed increased reactive oxygen species generation, up regulation of p53 protein expression, decreased survival markers level like p-Akt, p-NF-kB and p-STAT3 which were significantly higher after radiation treatment alone. We also found that combination treatment increased G2/M phase cell population, decreased IL-6, COX–2 and TNF-α expression and caused a loss in mitochondrial membrane potential with decreased level of angiogenesis marker MMP-9. Over expression of Bax and activation of caspase 3 indicated the apoptosis of the cells. The results provided a strong unique strategy to kill cancer cells HepG2, using less radiation dose along with effective pro-oxidant dose of EA

Stat-5A transfection confers resistance to CD4⁺ T cells from tumor-induced death.

<p>A, Stat-5A (<i>left panel</i>) and Stat-5B (<i>right panel</i>) isoforms were immunoprecipitated from cell lysates using specific antibodies and then Western blotted with anti-phospho-tyrosine or anti-Stat-5A/Stat-5B antibodies to determine phosphorylation status of specific proteins. B, Jurkat T cells were transfected with control vector, wild-type <i>Stat-5A/Stat-5B</i>, C-terminal truncated <i>Stat-5A</i>₇₁₃/<i>Stat-5B</i>₇₁₈ or constitutively active <i>Stat-5A1*6</i> genes and were cultured in the presence of media alone or MCF-7 spent media (±theaflavins) for 48 h. Percent cell death (Annexin-V-PE⁺/7AAD⁺) was determined flow cytometrically. Values are mean±S.E.M. of three independent sets of experiments.</p

Re-confirmation of PGE₂ as the molecule behind tumor-induced perturbation in CD4⁺ T cell survival signaling.

<p>A, MCF-7 cells were treated with theaflavins or celecoxib or transfected with Cox-2-siRNA and the levels of Cox-2 and GAPDH (internal control) mRNA were determined by RT-PCR (<i>upper panel</i>). Western blot analysis was performed for the determination of levels of Cox-2 or α-Actin (internal control) proteins (<i>middle panel</i>). In parallel experiments the amount of tumor-secreted PGE₂ in the cell-free supernatant was determined by ELISA (<i>lower panel</i>). B, Purified CD4⁺ T cells were cultured in the presence of media alone or MCF-7-spent media (tumors were either pre-treated with 25 µg/ml theaflavins/3.5 ng/ml PGE₂/50 µM celecoxib or transfected with 300pmole Cox-2-siRNA) for 48 h. Expression levels of IL2Rγc and Bcl-2 as well as phosphorylation status of Jak-3/-Stat-5 were determined by Western blotting in which α-Actin was used as internal control (<i>upper panel</i>). In parallel experiments, flowcytometric determination of percent cell death (<i>lower panel</i>) was established. Values are mean±S.E.M. of three independent experiments.</p

Tumor-shed PGE₂ is responsible for CD4⁺ T cell apoptosis.

<p>A, Tumor-secreted PGE₂ over time in cell-free spent media of MCF-7 cells (control (○), Cox-2-siRNA-transfected (Δ) or theaflavin-treated (•) was determined by ELISA. B, Percent CD4⁺ T cell death (Annexin-V-PE⁺/7AAD⁺), induced by the spent media as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007382#pone-0007382-g002" target="_blank">Fig. 2A</a>, was plotted over time. Values are mean±S.E.M. of three independent sets of experiments.</p

Cell free tumor supernatant leads to CD4⁺ T cell depletion by inducing apoptosis.

<p>A, Purified human peripheral CD4⁺ T cells were cultured in the presence of media alone or cell-free MCF-7-spent media (±theaflavins, doses from 6.25 µg/ml to 50 µg/ml). After 48 hours, viable cell numbers were scored by Trypan Blue exclusion method. B, Graphical representation of percent apoptosis of CD4⁺ T cells (<i>left panel</i>) and Jurkat T cells (<i>right panel</i>). CD4⁺ T cells labelled with Annexin V-PE and 7AAD were analyzed flow cytometrically. Annexin V/7AAD-positive cells were regarded as apoptotic cells. Values are mean±S.E.M. of five independent sets of experiments.</p