Sharma Sanjeev K, Yadav Subodh K, Sharma Ujjawal, Avti Pramod, Rana Satyavati, Khanduja Krishan L
Department of Biophysics, Postgraduate of Institute of Medical Education and Research, Chandigarh, India.
Department of Biotechnology, Maharishi Markandeshwar (deemed to be) University, Mullana, Haryana, India.
Anticancer Agents Med Chem. 2023;23(4):450-460. doi: 10.2174/1871520622666220527094219.
To find out the role of secretory phospholipase A2 (sPLA) isozymes as potential targets in tobacco condensate-induced colon damage.
The effects of cigarette smoke condensate (CSC) and the molecular mechanisms involved in the regulation of phospholipase A2 (PLA) and its isozymes in colon cells, which are still unclear and emerging, are studied.
The study aimed to check the effect of CSC on cell viability and reactive oxygen species (ROS) and superoxide. Also, the effect of CSC on gene expression of different secretory phospholipase A2 (sPLA) was evaluated. Moreover, the impact of inhibition of sPLA on various cell properties i.e. cell viability, cell proliferation, membrane damage and free radicals' generation is also studied.
CSC-induced changes were evaluated in cell viability by MTT assay, followed by the evaluation of membrane modulation by flow cytometry, free radical generation by fluorescent dyes, PLA isoforms gene expression patterns and their suppression by small interfering RNA (siRNA) studied in HCT-15 male and HT-29 female colon cells.
Our results demonstrate that HCT-15 and HT-29 cells treated with CSC significantly reduced the cell viability by 50% within 48 h and significantly enhanced the total reactive oxygen species (ROS) by 2 to 10-fold, and mitochondrial ROS (mtROS) and superoxide radicals (SOR) by 2-fold each. Treatment with CSC significantly unregulated secretory phospholipase A2 (sPLA) IID group and down-regulated IB and cytosolic phospholipase (cPLA) IVA groups in HCT-15 cells without affecting them in HT-29 cells. Silencing the sPLA IID group results in an increase in cell viability and a decrease in ROS. Silencing the PLA IVA gene in the HCT-15 cells showed a reduced expression which had no impact on the CSC-induced cell proliferation, membrane damage and free radicals (ROS, mtROS, and SOR) generation.
Therefore, identifying cell-specific sPLA isozymes seems to play a key role in controlling the ROSinduced damage by CSC and helps develop specific therapeutic strategies.
探究分泌型磷脂酶A2（sPLA）同工酶作为烟草冷凝物诱导结肠损伤潜在靶点的作用。
香烟烟雾冷凝物（CSC）的作用以及结肠细胞中磷脂酶A2（PLA）及其同工酶调控的分子机制仍不明确且正在研究中。
本研究旨在检测CSC对细胞活力、活性氧（ROS）和超氧化物的影响。此外，评估CSC对不同分泌型磷脂酶A2（sPLA）基因表达的影响。而且，还研究了抑制sPLA对各种细胞特性，即细胞活力、细胞增殖、膜损伤和自由基生成的影响。
通过MTT法评估CSC诱导的细胞活力变化，随后通过流式细胞术评估膜调节，通过荧光染料评估自由基生成，在HCT - 15雄性和HT - 29雌性结肠细胞中研究PLA同工型基因表达模式及其被小干扰RNA（siRNA）抑制的情况。
我们的结果表明，用CSC处理的HCT - 15和HT - 29细胞在48小时内细胞活力显著降低50%，总活性氧（ROS）显著增加2至10倍，线粒体ROS（mtROS）和超氧阴离子（SOR）各增加2倍。CSC处理显著上调HCT - 15细胞中分泌型磷脂酶A2（sPLA）IID组，下调IB和胞质磷脂酶（cPLA）IVA组，而在HT - 29细胞中对其无影响。沉默sPLA IID组导致细胞活力增加和ROS减少。在HCT - 15细胞中沉默PLA IVA基因显示表达降低，这对CSC诱导的细胞增殖、膜损伤和自由基（ROS、mtROS和SOR）生成无影响。
因此，确定细胞特异性sPLA同工酶似乎在控制CSC诱导的ROS损伤中起关键作用，并有助于制定特定的治疗策略。
