Antitumor Activity Evaluation And Mechanism Of Pyridine Substituted Derivatives At The C-17 Position Of Pregnenolone As Antiproliferative Agents In Vitro

To increase the selectivity of steroids and reduce side effects,the development of new compounds has been a challenge and has attracted widespread attention from medicinal chemists.Most chemotherapeutic drugs kill tumor cells through the apoptotic signaling pathway,and usually develop drug resistance.The study of necroptosis has become particularly important in cancer treatment.Because necroptosis can kill cancer cells by avoiding the resistance to apoptosis caused by cell arrest or escape.Our research group modified the structure of pregnenolone by introducing nitrogen-containing heterocyclic rings at the C-17 position in the early stage.In this thesis,the synthetic derivatives were evaluated by preliminary online activity prediction and antitumor activity screening in vitro,then the highly biologically active compound 2j was selected as the target compound to explore the anti-cancer mechanism of PC-3 cell.The specific research contents are as follows:1.Online software was used to evaluate the drug-likeness,biological activity,potential toxicity and related pharmacokinetic parameters of the derivatives.The results showed that most of the synthetic derivatives conformed to the three principles of the "Five Principles",a few of them met the four principles,and most of them had good biological activity,especially the ability to inhibit enzymes with the highest scores.All the compounds were safe and had no potential toxicity.Most of them had good absorption characteristic and high binding rate with plasma protein with almost no penetration to the blood-brain barrier.MTT test was used to screen the derivatives for preliminary anti-cancer activity in vitro.The results showed that the derivatives had certain cytotoxicity and selectivity to cancer cells,especially strong anti-proliferative ability against prostate cancer.Considering the above parameters,we finally selected 2j for further research.2.MTT and clone formation method were used to detect the proliferation inhibition effect of 2j on PC-3.The results showed that 2j inhibited PC-3 cell proliferation in time and concentration-dependent manner and also inhibited the formation of clones.Cell cycle detection kit was used to detect the effect of 2j on PC-3 cycle process.The results showed that 2j can induce PC-3 cell to be arrested at G0/G1 phase.The effect of 2j was detected by flow cytometry when Annexin V-FITC and PI stained PC-3 cell.The results showed that 2j can induce advanced apoptosis/necrosis of PC-3.Next,MTT and flow cytometry experiment were used to detect the effect of 2j on survival of PC-3 cell when 2j combined with caspase inhibitor or necroptosis inhibitor.The results showed that the death of PC-3 cell induced by 2j was caspase-independent,which can be partly blocked by Nec-1 inhibitor.Membrane rupture is a typical morphological feature of necroptosis.Therefore,we used trypan blue staining and LDH release experiment to detect the effect of 2j on the loss of membrane integrity and the activity of LDH when membrane ruptured.The results showed that some cells were stained blue by trypan.The integrity of cell membrane can be destroyed by 2j,and 2j can also increase the release of LDH,which can be reversed by Nec-1.Finally,Western blotting test was used to detect the expression of proteins related to necroptosis,and the results showed that 2j can up-regulate the key proteins of necroptosis.3.The effect of 2j on the migration ability of PC-3 was detected by scratch and Transwell method,and the expression of EMT-related proteins was detected by Western blotting test.The results showed that 2j can inhibit cell migration in vitro,down-regulate N-cadherin and Vimentin protein expression,and up-regulate the expression of E-cadherin.4.ATP detection kit was used to detect the effect of 2j on the ATP content in PC-3 cell.The results showed that 2j could reduce the ATP content.The active oxygen scavenger NAC was added to detect the effect of NAC on anti-tumor reversal.The results showed that NAC can weaken the cytotoxicity of 2j.The effect of 2j on intracellular ROS level in PC-3 cell was measured using an active oxygen detection kit.It was found that intracellular ROS level increased in a concentration-dependent manner,and then decreased after 12 h.Western blotting test was used to detect the expression of key proteins for necroptosis when 2j combined with NAC.We found that the combined use can reduce the expression of key proteins such as P-RIP1/3 and P-MLKL in the necroptosis signaling pathway.In summary,2j was selected as the target compound after evaluating the relevant parameters of the pregnenolone C-17 pyridine substituted derivatives and testing the antitumorr activity in vitro.It was determined that 2j can inhibit PC-3 cell proliferation,inhibit its migration in vitro,and induce necroptosis,mainly through the RIP 1/RIP3/MLKL signaling pathway.It may also be related to the production of reactive oxygen species.This research may provide experimental basis and lay theoretical foundation for the development of new steroidal antitumor drugs.