Cellular growth and the preparation of cells for division between two successive cell divisions is called the cell cycle. The cell cycle is divided into several phases; the length of these particular cell cycle phases is an important characteristic of cell life. The progression of cells through these phases is a highly orchestrated process governed by endogenous and exogenous factors. For the elucidation of the role of these factors, including pathological aspects, various methods have been developed. Among these methods, those focused on the analysis of the duration of distinct cell cycle phases play important role. The main aim of this review is to guide the readers through the basic methods of the determination of cell cycle phases and estimation of their length, with a focus on the effectiveness and reproducibility of the described methods.

LL-37, the only human cathelicidin that is released during inflammation, is a potent regulator of immune responses by facilitating delivery of oligonucleotides to intracellular TLR-9, thereby enhancing the response of human plasmacytoid dendritic cells (pDCs) to extracellular DNA. Although important for pathogen recognition, this mechanism may facilitate development of autoimmune diseases. In this article, we show that citrullination of LL-37 by peptidyl-arginine deiminases (PADs) hindered peptide-dependent DNA uptake and sensing by pDCs. In contrast, carbamylation of the peptide (homocitrullination of Lys residues) had no effect. The efficiency of LL-37 binding to oligonucleotides and activation of pDCs was found to be inversely proportional to the number of citrullinated residues in the peptide. Similarly, preincubation of carbamylated LL-37 with PAD2 abrogated the peptide’s ability to bind DNA. Conversely, LL-37 with Arg residues substituted by homoarginine, which cannot be deiminated, elicited full activity of native LL-37 regardless of PAD2 treatment. Taken together, the data showed that citrullination abolished LL-37 ability to bind DNA and altered the immunomodulatory function of the peptide. Both activities were dependent on the proper distribution of guanidinium side chains in the native peptide sequence. Moreover, our data suggest that cathelicidin/LL-37 is citrullinated by PADs during NET formation, thus affecting the inflammatory potential of NETs. Together this may represent a novel mechanism for preventing the breakdown of immunotolerance, which is dependent on the response of APCs to self-molecules (including cell-free DNA); overactivation may facilitate development of autoimmunity.

Three series of isomeric pyrrolo- and furo-fused 7-deazapurine ribonucleosides were synthesized and screened for cytostatic and antiviral activity. The synthesis was based on heterocyclizations of hetaryl-azidopyrimidines to form the tricyclic heterocyclic bases, followed by glycosylation and final derivatizations through cross-coupling reactions or nucleophilic substitutions. The pyrrolo[2',3':4,5]pyrrolo[2,3-d]pyrimidine and furo[2',3':4,5]pyrrolo[2,3-d]pyrimidine ribonucleosides were found to be potent cytostatics, whereas the isomeric pyrrolo[3',2':4,5]pyrrolo[2,3-d]pyrimidine nucleosides were inactive. The most active were the methyl, methoxy and methylsulfanyl derivatives exerting submicromolar cytostatic effects and good selectivity toward cancer cells. We have shown that the nucleosides are activated by intracellular phosphorylation and the nucleotides get incorporated to both RNA and DNA, where they cause DNA damage. They represent a new type of promising candidates for preclinical development toward antitumor agents.

In this work, we describe a new synthetic pathway to 30-methylidyne derivatives of lupane-triterpenoids that were consequently used as excellent substrates for the preparation of new compounds via Sonogashira coupling and via Cu^I catalyzed Azide-Alkyne 1,3-dipolar cycloadditions. Reactions had moderate to excellent yields. All prepared compounds were tested for their in vitro cytotoxicity on six cancer and two non-cancer cell lines. The most active compounds were triazoles with free 3-OH and 28-OH groups (betulin analogues) and among them, conjugate with triazole substituted by furan 16j was the best with IC₅₀ of 2.68 μM against CCRF-CEM and therapeutic index of 18.66. Mechanistic studies revealed that both 16g and 16j induce apoptosis in CCRF-CEM cells, as confirmed by annexin V/PI staining. Cell cycle analysis showed that 16j causes pronounced S phase arrest, while 16g modulates G1/S transition in a concentration-dependent manner. Moreover, 16g strongly suppressed DNA and RNA synthesis, whereas 16j paradoxically increased RNA synthesis despite replication inhibition. Both compounds triggered mitochondrial hyperpolarization, suggesting early mitochondrial involvement in their apoptotic mechanism. Western blot analysis supported these findings, revealing γH2AX induction and PARP cleavage, alongside distinct modulation of key regulators: 16j upregulated p21 and phospho-Chk1 (Ser345), while 16g downregulated both at high dose, consistent with checkpoint activation versus checkpoint bypass. These findings support 16g and 16j as promising candidates for further anticancer drug development.

A novel three-step synthetic approach for modification of betulin at position C-30 was developed starting from commercially available betulin diacetate. The scope of this procedure exceeds significantly the previously used methods while providing higher yields. The final Mitsunobu reaction was the pivotal step of the synthesis, and after optimization, 39 new derivatives─ethers, esters, and amines─were synthesized in good to high yields. All the novel compounds were tested for in vitro cytotoxic activity against six cancer cell lines and two noncancer cell lines. Compounds 30 and 37 show potent cytotoxicity against CCRF-CEM leukemia cells (IC₅₀ around 5 μM). In addition, 37 exhibits broad activity across multiple cancer cell lines, suggesting a promising multitargeted anticancer activity. Both compounds impair DNA and RNA synthesis, with 37 strongly inhibiting transcription at high doses. Analysis of apoptosis induction shows a divergent profile in both compounds; while 30 promotes robust apoptosis, derivative 37 appears to engage alternative cell death pathways. Biosynthetic disruption emerges as a promising anticancer strategy, with 37 as a top lead candidate.

The hedgehog signaling pathway plays an important role in vertebrate embryonic development, tissue homeostasis, and tumorigenesis. Constitutive activation of Hh signaling in various human tumors leads to GLI-mediated transcription and tumor progression. Based on the preliminary screening of a large library of known triterpenes that exhibited interesting Hh inhibitory activity, we designed and synthesized a new series of triterpenoid analogues containing aromatic heterocyclic substituents at position C-2 to enhance their interference with Hh signaling. In this study, we evaluated the effect of 15 synthesized triterpenoids on cell proliferation and Hh pathway activity in relevant cancer cell lines. Among these compounds, two derivatives, 11a and 11b, both featuring a furan ring at position C-2, demonstrated potent inhibitory effects on proliferation and induced cell death in nonsmall cell lung cancer (NSCLC) and prostate cancer cell lines exhibiting hyper-activated Hh signaling. Moreover, these compounds significantly reduced GLI-mediated transcription in cell-based reporter assays. Detailed immunoblot analyses revealed that compounds 11a and 11b decreased the expression of endogenous GLI1 protein and its target genes associated with tumor progression and proliferation, such as Cyclin D1, N-Myc, and Bcl-2, in A549 and DU-145 cancer cells. These findings suggest that the antiproliferative effects of 11a and 11b are mediated through inhibition of the Hh signaling pathway and are promising candidates for the development of new anticancer therapies targeting Hh-dependent tumors.