Ayse Tarbin Jannuzzi

Conventional pharmacological treatments aim to achieve homeostasis via specific drug interactions within target cells. However, this often results in cellular damage to non-targeted cells, which clinically manifests as adverse side effects. This is particularly significant in the case of chronic diseases for which curative treatment options are not available, dose increments are needed, and patients’ adaptive capacity is limited. To reduce such side effects, single-agent pharmacotherapy may involve the usage of partial agonists, selective receptor modulators (SERM), or pharmaceutical strategies designed to restrict drug distribution and spare sensitive tissues. However, in most cases, additive or synergistic combination therapies are needed to improve therapeutic efficacy and reduce adverse side effects. Historically, purified natural products paved the way for modern physiology and, later, pharmacology. This was followed by the revolutionary and efficient production of synthetic drugs, enabled by combinatorial chemistry, advances in molecular biology, and high-throughput screening. Today’s integrative approach favors a tendency toward the usage of natural drugs in drug combinations, not only as isolated natural molecules but also as complex plant extracts. These plant extracts consist of a combination of numerous chemical compounds that can have synergistic and antagonistic effects on specific biological processes. The growing number of ongoing and completed clinical trials across diverse disease settings continues to enhance our understanding of the efficacy and safety of natural product- drug combinations. This literature review offers a concise overview of the existing literature on these “synthetic-natural” pairings. By highlighting key preclinical findings and significant clinical trials, we explore how these hybrid strategies are poised to become the next frontier in medicine.

Hepatocellular carcinoma (HCC), the most common primary liver malignancy worldwide, is strongly associated with chronic Hepatitis B Virus (HBV) infection, a significant risk factor. The ubiquitin–proteasome system, central to protein degradation, cellular homeostasis, and cell cycle regulation, has been implicated in the pathogenesis of several cancers, including HCC. Despite this, the specific expression patterns of proteasomal subunits during HBV infection and HBV-induced HCC, as well as the association between mRNA expression of proteasomal subunits and proteasomal activity, remain poorly defined. To address this critical knowledge gap, we analyzed mRNA expression profiles of proteasomal subunits in HBV-infected humanized mouse models to uncover HBV-specific molecular alterations. Our findings revealed that the chymotrypsin-like activity (β5) subunit of the proteasome (PSMB5) is consistently overexpressed following HBV infection. Functional studies demonstrated that β5 deficiency decreases MHC I levels on the cell surface and leads to the accumulation of ubiquitinated proteins, establishing a direct link between β5 overexpression and increased proteasomal activity. Concordantly, HBV-infected patient livers—regardless of HCC status—displayed elevated β5 mRNA/protein levels and enhanced chymotrypsin-like activity. Additionally, analysis of Protein Atlas data revealed that elevated β5 mRNA expression correlates with poor clinical prognosis in HCC patients. In summary, this study highlights how HBV infection induces significant alterations in proteasome function by elevating β5 expression and activity in human and mouse livers. These findings underscore the critical role of proteasomal dysregulation in HBV-associated liver pathology and provide new insights into its involvement in HCC development. Understanding the interplay between HBV infection and proteasome dynamics offers a valuable avenue for the identification of novel therapeutic targets and biomarkers in HCC.

A series of aminated quinolinequinones linked to piperazine analogs (QQ1‐7) were synthesized and screened against the full panel of National Cancer Institute (NCI) cancer cell lines for their potential as cytotoxic agents. The Developmental Therapeutics Program of the NCI analyzed the NCI‐60 screening results and revealed that seven QQs were potent inhibitors of cancer cell growth across several cell lines, advancing them to the five‐dose assay. Encouraged by the NCI five‐dose assay results, the cytotoxicity of the selected QQs (QQ1 and QQ4) was further studied in three cancer cell lines—HCT‐116 (colon cancer), ACHN (renal cancer), MCF7, and T‐47D (breast cancer)—as well as in a normal cell line (HUVEC) for a deeper understanding. QQ1 was the hit compound for ACHN cells with an IC₅₀ value of 1.55 μM. QQ1 could inhibit ACHN cell proliferation, induce oxidative stress, and cause cell cycle arrest in ACHN cells. QQ1 did not affect the apoptotic value in ACHN cells. Oral bioavailability was poor for both QQ1 and QQ4 in vivo in rats due to faster intrinsic hepatic clearance in comparison with humans, as evidenced by in vitro metabolic studies with rat and human liver microsomes. Molecular docking simulation with putative target CDC25A revealed the interaction of QQ1 and QQ4 with active site residues responsible for substrate recognition.

The evaluation of in vitro biological activity of several previously reported quinolinequinones (AQQ1–5) against 60 human cancer cell lines (NCI-60) used by the National Cancer Institute’s Developmental Therapeutics Program (DTP) contributed to our earlier research on possible anticancer and/or antibacterial agents. Of interest, NCI-60 screening revealed that two quinolinequinones (AQQ1 and AQQ2) significantly reduced the proliferation of several cancer genotypes. Following the administration of a single dose and five additional doses, all quinolinequinones demonstrated a significant inhibitory effect on the growth of leukemia and other cancer cell lines. Hence, a series of subsequent in vitro biological assessments were performed to further understand the mechanistic impact of the compounds. In MTT assays, it was found that AQQ1 and AQQ2 exhibited higher efficacy against DU-145 cells (IC₅₀ 4.18 µM and 4.17 µM, respectively) compared to MDA-MB-231 (IC₅₀ 8.27 and 13.33 µM, respectively) and HCT-116 cells (IC₅₀ 5.83 and 9.18 µM, respectively). Additionally, AQQ1 demonstrated greater activity in this context. Further investigations revealed that AQQ1 inhibited DU-145 cell growth and migration dose-dependently. Remarkably, arrest of the DU-145 cell cycle at G0/G1 phase and ROS elevation were observed. Pharmacokinetic (PK) studies revealed that AQQ1 has better PK parameters than AQQ2 with %F of 9.83 in rat. Considering the data obtained with human liver microsomal stability studies, AQQ1 should have a better PK profile in human subjects. In silico studies (molecular dynamics) with three kinases (CDK2, CDK4, and MAPK) leading to cell cycle arrest at G₀/G₁ identified MAPK as a probable target for AQQ1. Taken together, our results showed that AQQ1 could be a potential chemotherapeutic lead molecule for prostate cancer.

Objectives: Glioblastoma is a fast-growing and aggressive brain tumor. Despite the current treatment methods, such as chemical and surgical operations, the prognosis is still poor. Therefore, combined therapeutic strategies are proposed to maximize therapeutic efficacy and reduce toxicity. Thymoquinone has been shown to have neuroprotective effects in addition to its anti-cancer effects on different types of cancer. 5-Fluorouracil, on the other hand, is a cytotoxic chemotherapy agent used to treat cancer. As a synergistic combinational approach, this study aimed to examine the antiproliferative effects and production of reactive oxygen species in a glioblastoma cell line. Methods: We have tested thymoquinone and 5-fluorouracil alone and in their combination to observe cellular growth with MTT assay. The combinational effects of the agents were determined by the CompuSYN software program. Cell proliferation was assayed with crystal violet assay. Reactive oxygen species production was analyzed by 2′,7′-dichlorodihydrofluorescein diacetate in glioblastoma cells. Results: Thymoquinone and 5-fluorouracil inhibited cell growth of glioblastoma cells with half maximal inhibitory concentrations (IC ₅₀ ) of 45.93 and 14.02 µM for 48 h, respectively. At synergistic combinational concentrations, the crystal violet assay demonstrated that there is a positive correlation between combination index values and cell proliferation. Also, an increment in the production of reactive oxygen species was observed upon combinational treatments. Conclusions: Our results indicate that the combinational strategy of these two agents reduced cell viability and proliferation in glioblastoma cells and showed strong synergistic anticancer efficiency.

It is quite challenging to find out bioactive molecules in the vast chemical universe. Quinone moiety is a unique structure with a variety of biological properties, particularly in the treatment of cancer. In an effort to develop potent and secure antiproliferative lead compounds, five quinolinequinones (AQQ1‐5) described previously have been selected and submitted to the National Cancer Institute (NCI) of Bethesda to envisage their antiproliferative profile based on the NCI Developmental Therapeutics Program. According to the preliminary in vitro single‐dose anticancer screening, four of five quinolinequinones (AQQ2‐5) were selected for five‐dose screening and they displayed promising antiproliferative effects against several cancer types. All AQQs showed a excellent anticancer profile with low micromolar GI₅₀ and TGI values against all leukemia cell lines, some non‐small cell lung and ovarian cancer, most colon, melanoma, and renal cancer, and in addition to some breast cancer cell lines. AQQ2‐5 reduced the proliferation of all leukemia cell lines at a single dose and five additional doses, as well as some non‐small cell lung and ovarian cancer, the majority of colon cancer, melanoma and renal cancer, and some breast cancer cell lines. This motivated us to use in vitro, in silico, and in vivo technologies to further investigate their mode of action. We investigated the in vitro cytotoxic activities of the most promising compounds, AQQ2 and AQQ3, in HCT‐116 colon cancer, MCF7 and T‐47D breast cancer, and DU‐145 prostate cancer cell lines, and HaCaT human keratinocytes. Concomitantly, IC₅₀ values of AQQ2 and AAQ3 against MCF7 and T‐47D cell lines of breast cancer, DU‐145 cell lines of prostate cancer, HCT‐116 cell lines of colon cancer, and HaCaT human keratinocytes were determined. AQQ2 exhibited anticancer activity through the induction of apoptosis and caused alterations in the cell cycle. In silico pharmacokinetic studies of all analogs have been carried out against ATR, CHK1, WEE1, CDK1, and CDK2. In addition to this, in vitro ADME and in vivo pharmacokinetic profiling for the most effective AAQ (AAQ2) have been studied.

With the development and approval of new proteasome inhibitors, proteasome inhibition is increasingly recognized in cancer therapy. Besides successful anti-cancer effects in hematological cancers, side effects such as cardiotoxicity are limiting effective treatment. In this study, we used a cardiomyocyte model to investigate the molecular cardiotoxic mechanisms of carfilzomib (CFZ) and ixazomib (IXZ) alone or in combination with the immunomodulatory drug dexamethasone (DEX) which is frequently used in combination therapies in the clinic. According to our findings, CFZ showed a higher cytotoxic effect at lower concentrations than IXZ. DEX combination attenuated the cytotoxicity for both proteasome inhibitors. All drug treatments caused a marked increase in K48 ubiquitination. Both CFZ and IXZ caused an upregulation in cellular and endoplasmic reticulum stress protein (HSP90, HSP70, GRP94, and GRP78) levels and DEX combination attenuated the increased stress protein levels. Importantly, IXZ and IXZ-DEX treatments caused upregulation of mitochondria fission and fusion gene expression levels higher than caused by CFZ and CFZ-DEX combination. The IXZ-DEX combination reduced the levels of OXPHOS proteins (Complex II–V) more than the CFZ-DEX combination. Reduced mitochondrial membrane potential and ATP production were detected with all drug treatments in cardiomyocytes. Our findings suggest that the cardiotoxic effect of proteasome inhibitors may be due to their class effect and stress response and mitochondrial dysfunction may be involved in the cardiotoxicity process.

Quinolinequinones, which are bicyclic heterocycles and quinone derivatives due to their broad range of biological activities, which include strong antifungal, antibacterial, antimalarial, and anticancer, have long been the subject of numerous investigations. After successful synthesis and characterization of aminated quinolinequinones (AQQ) and antimicrobial evaluation by our group, compounds were subjected to the NCI-60 Human Tumor Cell Lines Screen to determine anticancer activity. According to the NCI report, we further investigated the cytotoxic effects of selected compounds, AQQ6 and AQQ9, on the growth of DU-145 prostate cancer, MDA-MB-231 breast cancer, and HCT-116 colon cancer lines after 24 h treatment by MTT assay at 1–100 μM concentrations. HUVEC human umbilical vein endothelial cells were used as a non-cancerous cell line to determine the cancer selectivity of the compounds. Doxorubicin was used as a positive control drug. AQQ6 was more cytotoxic than AQQ9 and showed good cytotoxicity against DU-145 prostate cancer cells. Then, molecular pathways related to the cytotoxic effects of AQQ6 were investigated with analysis of cell cycle distribution, measurements of cellular ROS levels, and apoptosis/necrosis rate with flow cytometry at 1, 2.5, and 5 μM AQQ6 concentrations. According to our findings, AQQ6 induces G0/G1 cell cycle arrest dose-dependently. Additionally, apoptotic and necrotic cell populations significantly increased with 2.5 and 5 μM AQQ6 concentrations. AQQ6 did not affect cellular ROS levels. In conclusion, AQQ6 shows antiproliferative effects against DU-145 prostate cancer cells, which are mediated through cell cycle arrest and apoptotic and necrotic cell death. Potentially, AQQ6 can be a promising drug candidate for further anticancer research.

Plastoquinone analogs are privileged structures among the known antiproliferative natural product-based compound families. Exploiting one of these analogs as a lead structure, we report the investigation of the brominated PQ analogs (BrPQ) in collaboration with the National Cancer Institute of Bethesda within the Developmental Therapeutics Program (DTP). These analogs exhibited growth inhibition in the micromolar range across leukemia, non-small cell lung cancer (EKVX, HOP-92, and NCI-H522), colon cancer (HCT-116, HOP-92), melanoma (LOX IMVI), and ovarian cancer (OVCAR-4) cell lines. One brominated PQ analog (BrPQ5) was selected for a full panel five-dose in vitro assay by the NCI’s Development Therapeutic Program (DTP) division to determine GI₅₀, TGI, and LC₅₀ parameters. The brominated PQ analog (BrPQ5) displayed remarkable activity against most tested cell lines, with GI₅₀ values ranging from 1.55 to 4.41 µM. The designed molecules (BrPQ analogs) obeyed drug-likeness rules, displayed a favorable predictive Absorption, Distribution, Metabolism, and Excretion (ADME) profile, and an in silico simulation predicted a possible BrPQ5 interaction with proteasome catalytic subunits. Furthermore, the in vitro cytotoxic activity of BrPQ5 was assessed, and IC₅₀ values for U-251 glioma, MCF-7 and MDA-MB-231 breast cancers, DU145 prostate cancer, HCT-116 colon cancer, and VHF93 fibroblast cell lines were evaluated using an MTT assay. MCF-7 was the most affected cell line, and the effects of BrPQ5 on cell proliferation, cell cycle, oxidative stress, apoptosis/necrosis induction, and proteasome activity were further investigated in MCF-7 cells. The in vitro assay results showed that BrPQ5 caused cytotoxicity in MCF-7 breast cancer cells via cell cycle arrest and oxidative stress induction. However, BrPQ5 did not inhibit the catalytic activity of the proteasome. These results provide valuable insights for further discovery of novel antiproliferative agents.