Department of Botany, University of Calcutta, Kolkata, West Bengal 700019, India.
Department of Microbiology, University of Calcutta, Kolkata, West Bengal 700019, India.
J Integr Med. 2022 Sep;20(5):463-472. doi: 10.1016/j.joim.2022.06.004. Epub 2022 Jun 15.
"Multi-targeting" drugs can prove fruitful to combat drug-resistance of multifactorial disease-cervical cancer. This study envisioned to reveal if Thuja homeopathic mother tincture (MT) and its bioactive component could combat human papillomavirus (HPV)-16-infected SiHa cervical cancer cells since it is globally acclaimed for HPV-mediated warts.
Thuja MT was studied for its antiproliferative and antimigratory properties in SiHa cells followed by microscopic determination of reactive oxygen species (ROS) generation by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining and loss in mitochondrial membrane potential (MtMP) by rhodamine 123 (Rh123) staining. Apoptosis and autophagy inductions were studied by acridine orange/ethidium bromide (AO/EB) staining and immunoblot analyses of marker proteins. The bioactive component of Thuja MT detected by gas chromatography-mass spectrometry was studied for antiproliferative and antimigratory properties along with in silico prediction of its cellular targets by molecular docking and oral drug forming competency.
Thuja MT showed significant antiproliferative and antimigratory potential in SiHa cells at a 50% inhibitory concentration (IC) of 17.3 µL/mL. An increase in DCFDA fluorescence and loss in Rh123 fluorescence prove that Thuja MT acted through the burst of ROS and loss in MtMP respectively. AO/EB-stained cells under the microscope and immunoblot analyses supported Thuja-induced cellular demise via dual pathways-apoptosis and autophagy. Immunoblots showed cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) along with upregulation of Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B)-II, and p62 proteins. Hence, the apoptotic cascade followed a caspase-3-dependent pathway supported by PARP-1 cleavage, while autophagic death was Beclin-1-dependent and mediated by accumulation of LC3BII and p62 proteins. Thujone, detected as the bioactive principle of Thuja MT, showed greater anti-proliferative and anti-migratory potential at an IC of 77 µg/mL, along with excellent oral drug competency with the ability for gastrointestinal absorption and blood-brain-barrier permeation with nil toxicity. Molecular docking depicted thujone with the strongest affinity for mammalian target of rapamycin, phosphoinositide 3-kinase, and protein kinase B followed by B-cell lymphoma 2, murine double minute 2 and adenosine monophosphate-activated protein kinase, which might act as upstream triggers of apoptotic-autophagic crosstalk.
Robust "multi-targeting" anticancer potential of Thuja drug and thujone for HPV-infected cervical cancer ascertained its therapeutic efficacy for HPV infections.
“多靶点”药物在对抗多因素疾病——宫颈癌的耐药性方面可能是有效的。本研究旨在揭示土木香顺势疗法母酊剂（MT）及其生物活性成分是否能对抗人乳头瘤病毒（HPV）-16 感染的 SiHa 宫颈癌细胞，因为它在全球范围内被认可用于治疗 HPV 介导的疣。
研究了土木香 MT 在 SiHa 细胞中的抗增殖和抗迁移特性，然后通过 2'，7'-二氯二氢荧光素二乙酸酯（DCFDA）染色测定活性氧（ROS）的生成，通过罗丹明 123（Rh123）染色测定线粒体膜电位（MtMP）的丧失。通过吖啶橙/溴化乙锭（AO/EB）染色和标记蛋白的免疫印迹分析研究细胞凋亡和自噬的诱导。通过气相色谱-质谱法检测土木香 MT 的生物活性成分，研究其在 SiHa 细胞中的抗增殖和抗迁移特性，并通过分子对接和口服药物形成能力对其细胞靶标进行计算机预测。
土木香 MT 在 SiHa 细胞中的 50%抑制浓度（IC）为 17.3μL/mL 时，表现出显著的抗增殖和抗迁移潜力。DCFDA 荧光的增加和 Rh123 荧光的丧失证明土木香 MT 通过 ROS 的爆发和 MtMP 的丧失起作用。显微镜下 AO/EB 染色和免疫印迹分析支持土木香通过双重途径——细胞凋亡和自噬导致细胞死亡。免疫印迹显示半胱天冬酶-3 和多聚（腺苷二磷酸核糖）聚合酶-1（PARP-1）的切割，以及 Beclin-1、微管相关蛋白 1 轻链 3B（LC3B）-II 和 p62 蛋白的上调。因此，细胞凋亡级联反应遵循 caspase-3 依赖性途径，由 PARP-1 切割支持，而自噬死亡是 Beclin-1 依赖性的，并由 LC3BII 和 p62 蛋白的积累介导。检测到土木香 MT 的生物活性成分是侧柏酮，它在 IC 为 77μg/mL 时表现出更强的抗增殖和抗迁移潜力，并且具有良好的口服药物能力，具有胃肠道吸收和血脑屏障渗透的能力，且无毒性。分子对接显示侧柏酮与哺乳动物雷帕霉素靶蛋白、磷酸肌醇 3-激酶和蛋白激酶 B 的亲和力最强，其次是 B 细胞淋巴瘤 2、鼠双微体 2 和腺苷单磷酸激活蛋白激酶，这些可能作为细胞凋亡-自噬串扰的上游触发因素。
土木香药物及其生物活性成分土木香酮对 HPV 感染的宫颈癌具有强大的“多靶点”抗癌潜力，证实了其对 HPV 感染的治疗功效。
