Study on Leukamenin E induced cytoplasmic cytoskeleton rearrangement and migration inhibition in human cervical cancer HeLa cells
The microfilaments, microtubules, and intermediate fibers in eukaryotic cells are interconnected with their binding proteins to form a highly ordered three-dimensional network like cytoplasmic skeleton system. They participate in key cellular life processes such as material transport, information transmission, cell division, cell migration, and cell morphology maintenance. The dynamic process of the cytoskeleton is highly regulated to serve the needs of cellular life activities, and its assembly disorder can lead to cell growth inhibition, cell division arrest, and even cell apoptosis. Some anti-tumor drugs can disrupt the dynamic balance of microtubules by altering the depolymerization and polymerization of the cytoskeleton, thereby inhibiting cell proliferation and exerting anti-tumor effects. The plant derived anticancer drugs catharanthus alkaloids and taxanes can combine with β – tubulin, interfere with the dynamic process of microtubule skeleton, and be used to treat a variety of tumors, including breast cancer, lung cancer, neuroblastoma, rhabdomyosarcoma, acute leukemia, etc. The research and discovery of anti-tumor drugs targeting the cytoskeleton and its related proteins have become a very active frontier research field.

Reactive oxygen species (ROS) act as signaling molecules within cells and participate in major cellular life processes such as cell proliferation and differentiation. Due to changes in metabolic pathways in tumor cells, the content of ROS increases, especially H2O2, which makes tumor cells more sensitive to changes in ROS levels. Therefore, specific modifications of intracellular components by ROS generation and antioxidant defense have been identified as targets for cancer treatment. Research has shown that ROS generated by NADPH oxidase and other sources can directly modify microfilaments, microtubules, and intermediate silk proteins, affecting their dynamic assembly disassembly processes, or reshape the cytoskeleton by activating Rho GTPase upstream through related signaling pathways. NADPH oxidase mediated intracellular oxidative stress also plays an important regulatory role in cytoskeletal related diseases. In recent years, researchers have shown widespread interest in the involvement of ROS in the regulation of cytoskeleton dynamics.

There are over 90 species of plants in the Camellia genus distributed in China, of which about 30 are used for medicinal purposes. Among them, Isodon rubescens was once included in the Chinese Pharmacopoeia and is mainly used for the treatment of antibacterial, anti-inflammatory, and anti-tumor diseases. Oridonin, as the main active ingredient of Lonicera japonica, has been extensively studied for its anti-tumor mechanism, and its structural modifications have entered phase I clinical trials as anti leukemia drugs. More than 1300 species of enantiomeric kaempferol diterpenes have been discovered, particularly abundant in the branches and leaves of plants in the Camellia genus. These compounds exhibit extensive anticancer activity and are expected to lead to the discovery of new anti-tumor drugs. At present, only a very small number of enantiomeric diterpenes such as arbutin have been extensively and systematically studied for their anticancer mechanisms. There is still a lack of related research on other diterpenes of this type, but significant progress has been made in recent years. In recent years, it has been found that certain enantiomers of kaempferol diterpenes can act on the cytoskeleton system and exert anticancer activity. For example, bacteriocin A directly targets the BubR1 protein to activate the spindle assembly checkpoint, preventing mid to late cell transition and inhibiting the mitotic phase of leukemia cells Raji and Jurkat; Wangzaozin A and epinodosin induce rearrangement of the cytoskeleton (microfilaments, microtubules, and intermediate filaments) in HeLa and HL-60 cells, respectively, which is associated with their induction of cell differentiation; Leukamenin E can also induce keratin phosphorylation in HUVECs and PLC cells, thereby blocking the normal assembly of keratin fiber networks. This article continues to report the effects of leukamenin E on the rearrangement of three skeletal fibers (microfilaments, microtubules, and keratin fibers) in HeLa cells, as well as the possible mechanisms of cell growth and migration inhibition. It provides clues for elucidating the anticancer molecular mechanisms of enantiomeric kaempferol diterpenoids and scientific basis for the further development and application of this compound.

In recent years, the anticancer pharmacological activity of enantiomeric kaempferol diterpenes has attracted widespread interest among researchers. There have been increasing reports on the anticancer mechanisms of these compounds with different molecular configurations, and it has been found that their effects on cellular life processes are multifaceted, demonstrating multi-target effects at the molecular level. For example, inducing cell apoptosis and differentiation through various cellular signaling pathways; Targeting peroxidases I and II, BubR1 protein, and cancer protein AML1-ETO, it can regulate NADPH oxidase activity and induce keratin phosphorylation. Our research group has successively reported the effects of enantiomeric kaempferol diterpenes on the cytoskeleton system: leukamenin E can induce phosphorylation of cell keratin K8 and K18 and inhibit keratin assembly; Epinodosin and wangzaozin A can respectively affect the dynamic assembly of the cytoskeleton in HL-60 and HeLa cells, causing cytoplasmic cytoskeleton rearrangement, altering the distribution of microfilaments, microtubules, and intermediate fiber skeleton fibers, and interfering with the homeostasis of the cytoplasmic cytoskeleton system. However, their mechanisms of action are not well understood.

In this article, we first investigated the characteristics of HeLa cell cytoskeleton rearrangement induced by low concentrations of leukamenin E (0.4-1.0 μ mol/L). Low concentration of leukamenin E significantly changed the morphology of cells after prolonged treatment (48h and 72h), resulting in the formation of long stripes, a significant reduction in the number of pseudopodia, elongation of pseudopodia, and a significant change in nuclear morphology, with an increase in the number of “kidney shaped nuclei”. As is well known, microfilaments, microtubules, and medium fibers all have the function of maintaining the morphology of cells and their nuclei. Therefore, these changes suggest that leukomenen E may interfere with the dynamic balance of the cytoskeleton and induce rearrangement of the cytoskeleton. Further fluorescence staining confirmed this speculation: the number of cytoplasmic stress fibers decreased, microtubules and keratin fibers gathered around the nucleus, the arrangement of microtubule and keratin intermediate fibers changed significantly, and some microtubule and keratin intermediate fibers thickened. Flow cytometry analysis also confirmed a decrease in cohesive microfilaments, a significant increase in aggregated microtubules, and a slight increase in keratin fiber polymerization in HeLa cells. This result is similar to our previous report on the induction of cytoskeleton rearrangement in HeLa cells by the enantiomeric diterpenoid wangzaozin A. However, treatment of HepG2, H1299, PLC, and HUVEC cells with leukamenin E at high concentrations (2.0~4.0 μ mol/L) for 24 hours resulted in minimal changes in cell morphology, which is completely different from the pattern of changes observed after low concentration treatment. We found that high concentrations of leukomenen E can activate the ERK signaling pathway in cells, induce phosphorylation of K8-S431/73 and K18-S52, inhibit keratin fiber assembly, and reduce the number of keratin fibers. This indicates that different concentrations of leukomenen E can participate in regulating different signaling pathways in cells, causing different cellular response events.

The deep involvement of the cytoskeleton in cell division and migration is closely related to cellular carcinogenesis and deterioration. The dynamic fiber network composed of the cytoskeleton and regulatory proteins is involved in the process of cell migration, manifested as continuous changes in cell morphology, reorganization of the cytoskeleton, and driving directional cell movement. Compounds targeting the cytoskeleton, such as cytochalasin, colchicine, and vincristine, inhibit cell proliferation and migration by affecting the assembly of the cytoskeleton. Our research shows that 0.8~1.0 μ mol/L leukomenen E can interfere with the dynamic balance of HeLa cell cytoskeleton, causing significant rearrangement of cytoplasmic cytoskeleton, inhibiting cell migration, and reducing cell proliferation rate by blocking cell cycle operation. This indicates that leukomenen E has important value in the research and discovery of new anti-cancer drugs.

ROS is a type of oxygen-containing free radical produced in living organisms that is more active than molecular oxygen chemistry, such as O2, H2O2, · OH, etc. Under normal physiological conditions, ROS plays an important role as a second messenger in determining cell fate and modifying various signaling molecules. Research has confirmed that ROS participates in the signaling pathway of cytoskeleton remodeling, directly modifying the cytoskeleton or proteins related to the cytoskeleton, and is an important regulatory factor involved in the structure and function of the cytoskeleton. Previously, we reported that leukomenen E can increase intracellular ROS levels and induce HL-60 cells to differentiate into mature granulocytes by regulating NADPH oxidase activity in HL-60 cells. In this study, treatment of HeLa cells with 0.4~1.0 μ mol/L leukomenen E significantly increased intracellular ROS levels, and ROS content significantly increased with increasing drug concentration and prolonged treatment time. We speculate that ROS may be an upstream event of leukomenen E-induced cell morphological changes, cell migration inhibition, and cytoskeleton rearrangement. Leukmenen E may regulate cytoskeleton rearrangement through the ROS signaling pathway, leading to changes in cell morphology and inhibition of cell migration. Further experiments confirmed the above speculation: the addition of NAC has a significant inhibitory effect on the morphological changes induced by leukomenen E (towards elongated and reduced elongated pseudopodia), and it was observed that NAC weakened the induction of stress fiber reduction by leukomenen E and reduced the aggregation of microtubules and keratin fibers around the nucleus. These phenomena indicate that the rearrangement of the cytoskeleton and changes in cell morphology caused by leukomenen E are associated with an increase in ROS levels. Studies have shown that ROS is involved in the dynamic processes of microfilament aggregation and depolymerization. In human primitive chondrocytes, an increase in ROS promotes oxidative modification of Racl and induces changes in the cytoskeleton structure of actin cells; ROS disrupts the stress fiber network and other microfilament bundles of testicular Sertoli cells (SC) through the ERK pathway. In addition, ROS significantly affects the dynamic instability of microtubules, regulates microtubule protein cytoskeleton organization, and induces microtubule protein modification. There are no relevant reports on the correlation and mechanism between ROS and keratin fiber polymerization. We speculate that the rearrangement of the cytoskeleton caused by leukamenin E is triggered by the activation of signaling pathways related to cytoskeleton rearrangement through the increase of ROS levels, but further research is needed to confirm the specific pathway. Lai et al. reported that naringin induced an increase in ROS levels in HeLa cells, activated the ROS/JNK/Bcl2 pathway, and inhibited cell proliferation and migration. Our research confirms that ROS can act as signaling molecules on the cytoskeleton system, interfering with the coordinated reorganization of the cytoskeleton during cell movement, leading to inhibition of cell migration. Further in-depth research is needed on the signaling pathway through which leukamenin E inhibits cell proliferation.