Additionally, we introduce a novel approach to derive an approximate value of the MCF MKM model parameters by knowing only the animal species and the mean number of chromosomes. The overall good agreement between MCF MKM predictions and in vitro data suggests the MCF MKM can be reliably used for the RBE calculations. In most cases, a reasonable agreement was found between the MCF MKM and the LEM IV.The relative biological effectiveness (RBE) calculations used during the planning of ion therapy treatments are generally based on the microdosimetric kinetic model (MKM) and the local effect model (LEM). The Mayo Clinic Florida MKM (MCF MKM) was recently developed to overcome the limitations of previous MKMs in reproducing the biological data and to eliminate the need for ion-exposed in vitro data as input for the model calculations. Since we are considering to implement the MCF MKM in clinic, this article presents (a) an extensive benchmark of the MCF MKM predictions against corresponding in vitro clonogenic survival data for 4 rodent and 10 cell lines exposed to ions from  The moddel LQM ,18) are  are ß) odel LQM ,19. . ß of thWhile amorphus track structure models such as the LEM  rely on Despite some limitations of the LEM I  were repß) of the LQM [The original version of the MKM , develop the LQM ,11,31. ß by using whole spectral information. Additionally, the mean radius of the radiation-sensitive subnuclear domains is a priori calculated using a new methodology based on the population-mean amount of DNA content in the cell nucleus [Rn, mean radius subnuclear domains rd), the MCF MKM was successful in predicting average and experiment-specific RBE trends for the most commonly used mammalian cell line  and the clinically relevant human salivary gland tumor cells (HSG cell line) for ions from 1H to 132Xe. A sensitivity test proved that the a priori determined MCF MKM parameters for these two cell lines were in good agreement with the ones obtained by fitting the in vitro data [In view of the upcoming integrated proton-carbon ion therapy center at Mayo Clinic Florida , a nove nucleus . This istro data . 1H to 238U. Since morphologic measurements of the cell nucleus (Rn) are not always available in the literature, this work also presents a phenomenological approach to derive an approximate value of both model parameters  by knowing the animal species and, for aneuploid cancer cells, the mean number of chromosomes. When possible, the MCF MKM prediction were compared with published results of the LEM IV [Considering a possible clinical implementation of the MCF MKM, it is of primordial importance to (a) validate the model predictions against a large dataset of in vitro clonogenic survival data for various cell lines and (b) compare the MCF MKM results with the results of the most recent version of clinically relevant models such as the LEM. Therefore, in this article we investigate the accuracy of the MCF MKM in systematically predicting the LQM terms and the RBE values for 4 rodent  and 10 human repair-competent cell lines in case of exposures to ions from e LEM IV . ß) and the RBE for a surviving fraction of 10% (RBE10%) predicted by the MCF MKM are compared with the experimental in vitro data from PIDE 3.2 [1H and 2H ions) were pooled together since negligible differences were observed when the results are plotted as a function of the LET [In the following paragraphs, the linear and quadratic terms of the LQM (α and PIDE 3.2  and publPIDE 3.2  with thePIDE 3.2 ). Since  the LET ,36.10% for better clarity in the comparison between the three data series . Nonetheless, examples of RBE calculations for other surviving fractions (50% and 1%) are given in Though the MCF MKM can be used to compute the RBE also for other surviving fractions, the plots are limited to the RBEß [The PIDE in vitro database does not provide uncertainty intervals for the clonogenic survival data. This is due to the fact that most of the published biological studies do not include an uncertainty analysis and, in some cases, not even an indication of the statistical dispersion of the results. The RBE results for the clonogenic survival essay are affected by several sources of uncertainty, such as: the different operational protocols between different groups , cell lß ). All ofß , is to bß ,36) and ß . Consequ1H ions where the LEM IV seems to predict higher α and RBE10% values, the results of the calculations with the two models are in good agreement between each other and with the in vitro data of 1H to 20Ne). It is worth remembering that the prediction of large ß values  is beyond the capabilities of both models. Furthermore, it should be noted that, here and in the following, the ß values predicted by the LEM show a sharper decrease with the increase of the LET with respect to the ß values calculated with the MCF MKM. In case of 28Si and heavier ions  are shown in ier ions , the agr40Ar ions. In this case, the MCF MKM predictions appear to be higher than the in vitro data. No LEM IV calculations for the CHO cell line were available for 40Ar ions. The somehow counterintuitive increase of the LEM-calculated ß term at very high LET is due to the current implementation of the DNA damage enhancement factor [The MCF MKM and the LEM IV results appear to be in good agreement with the in vitro data for chinese hamster ovary cells (CHO and CHO-K1 cell lines) over the whole investigate ion-LET range . The onlt factor .1H and 7Li ions. Except for the larger in vitro ß data point for 26 keV/µm protons , the model calculations  are in good agreement with the in vitro data for both 1H and 7Li ions. Due to the approach used in Equation (4), the MCF MKM always predicts a monotonic decrease of ß with the increase of the particle LET . The MCF MKM predictions for transformed mouse epidermal cells (PDV cell line) are plotted in ß value around 160 keV/µm, this has a little effect on the calculated RBE10% since the α term is significantly larger than ß. As for α, the RBE10% calculated with both models are similar and in good agreement with the experimental data.The α values predicted by MCF MKM and the LEM IV for carbon-irradiated rat prostatic adenocarcinoma epithelial cells  overestimate the in vitro α values for 1H ions. The overestimation is significantly larger for the LEM IV. By contrast, the proton ß values calculated by the two models agree well between each other but underestimate the in vitro ß data. This concomitant overestimation of α and underestimation of ß might be an artifact due to the anti-correlation between the two LQM terms during the fit of the in vitro data [10% values predicted by the MCF MKM are in good agreement with the corresponding in vitro results for 1H ions. By contrast, the LEM IV seems to overestimate the HeLa proton RBE10%. Though the results of the three data series  are in closer agreement, the discussion of the HeLa results for 4He ions follows the one for 1H ions and it is not repeated. The results of human cervical cancer cells (HeLa cell line) are plotted in tro data ,45. None10% calculated by the two models (MCF MKM and LEM IV) in case of proton-irradiated human fetal lung fibroblasts (HF19 cell line) are higher than the corresponding in vitro data . 12C ions. No LEM IV data were available. A good agreement between the calculated and the measured cell response is present over the whole LET range. The MCF MKM results for human mammary epithelial cells (M/10 cell line) are shown in 12C, 20Ne, and 28Si ions. For 12C ions, the α and RBE10% values calculated with the MCF MKM and the LEM IV are reasonably similar and in good agreement with the in vitro data. The MCF MKM accurately describes the α and the RBE10% trends for 20Ne ions. The α and RBE10% values calculated by the MCF MKM for 28Si ions appear to underestimate the in vitro data. The LEM IV appears to systematically underestimate α and RBE10% for both 20Ne and 28Si ions. Once the dispersion in the in vitro ß values for 12C and 20Ne ions is considered, we conclude that both models describe this quantity reasonably well. On the other hand, though the ß values calculated by the MCF MKM and LEM IV for 28Si ions are somehow similar, both models significantly overestimate the experimental trend of ß.1H ions, the MCF MKM and the LEM IV seem to overestimate the in vitro α and RBE10% values. The overestimation is larger for the LEM IV. Except for the two highest-LET in vitro entries for 12C ions, the MCF MKM seems to well reproduce the in vitro α values. On the other hand, the LEM IV results appear to be smaller. However, the LEM IV seems to better describe the ß trend as a function of the LET for 12C ions. The in vitro RBE10% of 12C with LET < 100 keV/µm appears to be underestimated by both models, with the LEM IV showing the smaller RBE10% values. The MCF MKM seems to reproduce the in vitro α entry for 40Ar ions reasonably well. Though larger than the corresponding in vitro entry, both models predict a similar ß value for 40Ar ions. The MCF MKM and the LEM IV, respectively, overestimates and underestimates the RBE10% for 40Ar ions.The in silico and in vitro data for human laryngeal squamous cell carcinoma (SQ20B cell line) are compared in 4He, 12C, 20Ne, and 28Si ions) and 40Ar, 56Fe, and 238U ions). In case of 4He ions, the MCF MKM seems to accurately reproduce the in vitro data. By contrast, the LEM IV appears to overestimate the α and RBE10% in vitro data for 4He ions. For ions from 12C to 56Fe, the in vitro data are characterized by large values of ß  which are underestimated by both models. Therefore, due to the previously discussed anti-correlation between α and ß [10% values computed with the MCF MKM are in satisfactory agreement with the in vitro results for these ions (12C to 56Fe). Interestingly, the α values calculated by the LEM IV for ions from 12C to 56Fe agree reasonably well with the in vitro data. However, the RBE10% predicted by the LEM IV are systematically smaller than the in vitro data (12C to 56Fe ions). In case of 238U ions, the results of both models are in reasonable agreement with the in vitro data.The results of human kidney cells (T1 cell line) are plotted in  α and ß ,45, it w10% results of the LEM IV are systematically lower than the corresponding in vitro ones. The spread in the in vitro ß data prevents from drawing any conclusion on which model better describes this quantity for the TK1 cell line. As can be seen in 10% values calculated by both models are similar and in good agreement with the in vitro data. ref (=0.031 Gy−1), an unusually low αref/ßref ratio (=0.56 Gy), and relatively large in vitro values of α for the 12C ions exposures (>0.4 Gy−1). Therefore, the in vitro RBE for low dose exposures (RBEα = α/αref) shows very large values  are compared to the carbon-exposed in vitro data in The linear-quadratic model  ,18) was S is the surviving fraction as a function of the absorbed dose D, α and ß are exposure- and cell- specific fitting parameters.S (RBES) was calculated using Equation (2) [ß, αref and ßref are the linear and quadratic terms of the LQM for the radiation under investigation and the reference photon exposure respectively.The RBE for the surviving fraction tion (2) (2)RBES=All in vitro clonogenic survival data were extracted from the Particle Irradiation Data Ensemble (PIDE ) versionThe following physical information are listed for each entry: particle type, energy, LET in water, beam type (monoenergetic or a spread out Bragg peak), cell line details , and the cell cycle phase. Two sets of linear-quadratic terms are included in PIDE: the result of the fit performed by the authors of the original publication and by the PIDE team. When possible, the second set of LQM terms was preferred due to the systematic fit procedure by the PIDE team ,35). Uncref and ßref values. Similarly, we included PIDE entries for which at least one of the two LQM sets has a non-negative value of ß for the ion exposure. These negative ß values are supposed to be due to the presence of subpopulations of cells with different radiation resistance [ß. This initial filtering of PIDE led to a database containing 629 entries: 296 for human cell lines and 333 for rodent cell lines. For consistency with the in silico calculations performed with monoenergetics ions, we discarded PIDE entries for cell exposures within spread out Bragg peaks. To avoid partial cell irradiations and the large uncertainties associated with very-low energy exposures, we discarded entries in case of ions with energy <1 MeV/n. Afterwards, since this work deals with asynchronized repair-competent cell lines only, we included only asynchronized PIDE entries for which at least one of the two LQM sets  includes positive αsistance . HoweverThe following paragraphs describe the process of in vitro data selection for both rodent and human cell lines. An overview on the cell lines included in this study  is given in After the initial filtering, 333 PIDE entries were available for rodent cell lines. Since the results of Chinese hamster lung fibroblasts (V79 cell line) were topic of a previous investigation [40Ar and heavier ions) with energy less than 10 MeV/n. However, in case of ions heavier than 20Ne, it was only possible to accurately compute the microdosimetric spectra for ions of energy equal or greater than 10 MeV/n (see 85 of the remaining 122 PIDE entries were in case of repair-competent Chinese hamster ovary cells (CHO and CHO-K1 cell lines), Chinese hamster peritoneal fibroblasts (B14FAF28 cell line), and mouse embryo fibroblasts (C3H10T1/2 cell line). All the B14FAF28 entries in PIDE were extracted from a single study  and are eV/n see . TherefoThe remaining 37 rodent entries in PIDE were for 16 different cell lines. Cell lines with less than 3 entries for at least one ion were discarded. The remaining 14 PIDE entries were in case of rat intestinal epithelial fibroblasts (IEC-6 cell line), rat prostatic adenocarcinoma epithelial cells (R-3327-AT-1 cell line), epidermal cells of a newborn mice transformed with the carcinogenic dimethylbenzanthracene (PDV cell line), and squamous skin cancer cells obtained by injecting PDV cells in mice (PDV C57 cell line). Since the number of chromosomes and the radiosensitivity of the IEC-6 cell line were reported to significantly vary with the passage number , this ceref/ßref ratio of ~0.6 Gy, and the availability of morphologic and karyotypic information in the literature. The 8 cell lines with most entries in PIDE were: kidney cells (T1 cell line), skin fibroblasts (NB1RGB cell line), laryngeal squamous cell carcinoma (SQ20B cell line), myeloid leukemia cells (TK1 cell line), leukemia cells (HL-60 cell line), fetal lung fibroblasts (HF19 cell line), mammary epithelial cells (M/10 cell line), and glioblastoma cells (U-87 cell line). Entries for U-87 MG cell line were not pooled together with the ones for the U-87 cell line because of their different radiosensitivity , glioblastoma cells (A-172 cell line), and foreskin fibroblasts (AG01522 cell line) were excluded since part of other MCF MKM studies  are calculated using Equations (3) and (4) respectively.The MCF MKM  was usedc(y) accounts for the non-Poisson distribution of lethal lesions at high LET and is calculated with Equation (5).The correction factor d(y) is the dose probability density of the lineal energy y, α0 and ß0 are the LQM terms in the limit of y → 0, rd is the mean radius of the subnuclear domains, and ρ is the density (=1 g/cm3). ß  can be computed using a quadratic implementation (Equation (4)) of the correction factor c(y) (Equation (5)) used for the calculation of α [The equation used to assess α (Equation (3)) is an alternative implementation of the non-Poisson MKM . While tion of α .Rn), the mean radius of the spherical subnuclear domains (rd), the LQM terms for the reference photon exposure (αref and ßref), and the LQM terms in the limit of y → 0 (α0 and ß0). No in vitro ion-exposure data were used at any point for the assessment of these parameters. All the model parameters are representative of population-mean characteristics [Rn, rd, αref and ßref used in the MCF MKM calculations are listed in In addition to the simulated microdosimetric spectra , the MCFeristics  and are rd and, in some cases, also of Rn: the mean DNA content of the irradiated population p is the ploidy number, and ξ is a factor accounting for the cell cycle distribution of the irradiated population , p is ted cells ).p is equal to 2 for healthy cell lines and for diploid cancer cells. Though mutated chromosomes might contain an abnormal amount of DNA, an approximated value of p for aneuploid cancer cells can be calculated with Equation (7)x is the mean number of chromosomes in the aneuploid cell population and xn is the number of chromosomes in a normal set .p was set to 2 for the healthy cell lines  and for the myeloid leukemia cells (TK1 cell line) which were reported to be pseudodiploid [x was extracted from literature and it is equal to: 78 for the HeLa cell line [In this work, the ploidy number odiploid . For theell line , 44 for ell line , 62 for ell line , 59 for ell line , 39.4 foell line , and 54.ell line . Since well line .Rn. Details of the morphological information from literature can be found in Three approaches are used for the assessment of the mean radius of the cell nucleus (1) Preferably, the radius of the cell nucleus is extracted from literature as determined by means of morphologic measurements for cells presenting a spherical nucleus . This is the approach used in our previous work  dealing However, since this information is not always available, two additional strategies are used in this article. Rn) can be assessed from the cross-sectional area of the nucleus measured in case of attached cells. Generally, Rn is calculated with Equation (8)A is the cross-sectional area of the nucleus. This approach implicitly assumes that the half-thickness of the nucleus is equal to the radius assessed with Equation (8). However, the nucleus of cells attached during the microscopic measurements is not spherical, but more similar to an oblate spheroid/ellipsoid. Published results indicate that the vertical semi-axis of the oblate spheroid/ellipsoid is roughly a third of the mean radius of the cross-sectional area of the nucleus [(2) The mean radius of the spherical nucleus  and the minor semi-axis equal to a third of the equatorial radiusA is the cross-sectional area of the nucleus.Thus, we introduce an alternative formula (Equation (9)) to calculate Γ and the mean radius of the cell nucleus Rn for asynchronized cells. In this regard, Rn plotted as a function of the mean DNA content of 22 different asynchronized cell lines  If also no cross-sectional area measurements are available, we propose a novel phenomenological correlation between the mean DNA content of an asynchronized population cal data ,67,68,69The subnuclear domains in MKMs are meant to represent the dimension of subnuclear structures where the accumulation of radiation-induce damage (likely DNA DSBs) is thought to correlate with cell death ,26,70. Prd) can be assessed with Equation (11) [Therefore, under the simplifying assumption that the DNA is homogenously distributed within the cell nucleus, we hypothesize that the MCF MKM subnuclear domains represent the average volume of the cell nucleus containing 2 Mbp of DNA. Thus, the mean radius of the subnuclear domains (ion (11) :(11)rd=Rref and ßref are simply the α and ß values for that reference photon survival curve. Alternatively, as done in previous studies [ref and ßref were calculated with Equations (12) and (13):ref,i and ßref,i are the LQM terms for a reference photon exposure, and ni is the number of ion-exposure entries for that set of αref,i and ßref,i. This weighted sum was used to prevent photon experiments with few ion-exposure entries from biasing the calculation of the average αref and ßref. This approach was used for half of the cell lines included in this article, namely: CHO and CHO-K1, C3H10T1/2, HF19, NB1RGB, SQ20B, T1, and TK1.If all PIDE entries for a specific cell line share the same photon reference irradiation, then α studies ,33, in s0 and ß0 are determined by analyzing only photon in vitro data. Since the integral in Equation (5) is equal to ~1 for the reference photons, then ß0 = ßref. α0 can be calculated using Equation (14) [Similarly, αion (14) :(14)α0=αThe dose-mean lineal energy 60Co and 137Cs) and energetic X-rays (6 MV), 4.3 keV/µm for less energetic X-rays  [Since it is unpractical nor feasible to perform experiment-specific calculations of  X-rays) .1H, 4He, 7Li, 12C, 16O, 20Ne, 28Si, 40Ar, 56Fe, 238U ions were simulated. The energy of the ions was equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 MeV/n. The lineal energy spectra for ions heavier than 20Ne with energy lower 10 MeV/n were disregarded because of the presence of an inappropriate peak . A logarithmic binning from 10−2 to 107 keV/µm with 50 bins per decade was used to score the lineal energy spectra. The minimum energy deposition considered in the microdosimetric calculations was that for one event of one ionization only (10.9 eV [The computation of the microdosimetric spectra and the LET follows the methodology of our previous study with the MCF MKM  and only(10.9 eV ). http://web-docs.gsi.de/∼weick/atima/ (accessed on 5 September 2022) implemented in PHITS for the beams used to assess the microdosimetric spectra. Since the monoenergetic simulations were performed over an infinitesimal target, the simulated track- and dose- mean LET values were equivalent. By contrast, the in vitro experimental exposures are performed with quasi-monoenergetic beams. Consequently, track- and dose- mean LET values could differ. The inclusion of secondary fragments in the mean LET calculation and the use of different methods  significantly influence the LET results. Since details on the LET calculations were not always included in the publications from which the in vitro data were extracted and because different articles used different methodologies to calculate and average the LET, all results of this study are plotted as a function of a generic “unrestricted LET in water”.In order to facilitate the comparison between the in silico and in vitro data, the unrestricted LET in water was assessed using the ATIMA model  suggests that the MCF MKM can accurately predict the in vitro clonogenic survival without requiring ion-irradiated in vitro data as input. This was possible thanks to several new strategies introduced to a priori determine the MCF MKM parameters by means of morphologic and karyotypic information.The systematic benchmark performed in this work  and the use of average radiosensitivity predictions for the MCF MKM , a reasonable agreement between the results of the models was found in most cases.Future work with the MCF MKM is planned to include in the model dose-rate and fractionation effects, enhanced cell radioresistance due to hypoxia, and the transition of the survival curve to a constant slope at high doses. Additionally, we will investigate the possibility to extend the MCF MKM predictions to in vivo and patient data.


Oncological diseases have currently reached an epidemic scale, especially in industrialized countries. Such a situation has prompted complex studies in medicinal chemistry focused on the research and development of novel effective anticancer drugs. In this review, the data concerning new 4-thiazolidinone-bearing hybrid molecules with potential anticancer activity reported during the period from the years 2017–2022 are summarized. The main emphasis is on the application of molecular hybridization methodologies and strategies in the design of small molecules as anticancer agents. Based on the analyzed data, it was observed that the main directions in this field are the hybridization of scaffolds, the hybrid-pharmacophore approach, and the analogue-based drug design of 4-thiazolidinone cores with early approved drugs, natural compounds, and privileged heterocyclic scaffolds. The mentioned design approaches are effective tools/sources for the generation of hit/lead compounds with anticancer activity and will be relevant to future studies. Cancer diseases are a colossal problem for humanity, and according to data from the World Health Organization (WHO), they are the major cause of death among people under 70 years . This poMolecular-hybridization approaches are important synthetic strategies in the design of drug-like small molecules with more powerful and biologically more extensive anticancer properties than the initial compounds .4-Thiazolidinone scaffolds belong to privileged structures in drug design  and glit4-Thiazolidinone scaffolds are widely used for the development of anticancer agents, and a number of recent reviews have been published dedicated to this area of drug design ,10. The A series of publications reported the successful application of molecular hybridization methods and strategies for the design of novel anticancer agents based on obtaining hybrids of the type “approved drugs-4-thiazolidinone scaffolds”. The drugs from different pharmacological groups were used as parent molecules for further hybridization and development.1–3, were identified. Compounds 1–3 induced apoptotic cell death. Moreover, cycle arrest in the G0/G1 phase was caused by compounds 2 and 3, while compound 1 inhibited the cell cycle in the G2/M phase. Hybrid molecules 1 and 3 proved to have stronger genotoxic effects than imatinib against K562 cells [Türe et al.  reported62 cells .Recent decades have been marked by an increased interest in the use of potential NSAIDs for the treatment of cancer or using NSAIDs scaffolds/molecules for the design of new antimitotic agents . 4 and 5, were found to be efficient in some types of cancer compared to the effect of doxorubicin (reference drug), with the highest activity level detected on the MCF7 line, with IC50 values of 0.31 and 0.30 µM, respectively. Furthermore, tumor specificity and minimal effects on normal fibroblasts were observed for 4 and 5. In vitro studies of the molecular mechanisms of action for 4 and 5 revealed the induction of DNA damage, cell cycle arrest in the G2/M phase, and the induction of apoptosis as indicated by annexin-V staining and the activation of caspases. Moreover, it was found that both compounds modulate the expression and activity of several factors in the DNA damage response pathway, cyclins/cyclin-dependent kinases, and CDC25 phosphatase.Ramadan W.S. et al.  used a h6 and 7  inhibitors by Eldehna et al. . The new9  was foun10 -2-chloro-3-(4-nitrophenyl)prop-2-enal (CAS 3626-97-9) and was used as a drug in medical practice in the former Soviet Union , and LC3B (the microtubule-associated protein 1A/1B light chain 3B), suggesting its influence on the autophagy process.The diversity-oriented approach was applied for the synthesis of ciminalum-4-thiazolidinone hybrid molecules, and a series of derivatives were reported by Buzun and Finiuk ,25,26. F11  was cyto12 [50 = 1.57 µM and TGI = 13.3 µM). The analysis of activity inside cancer panels revealed a certain sensitivity profile in the GI50 concentration range of < 0.01–0.02 µM toward leukemia , colon cancer (SW-620), CNS cancer (SF-539), and melanoma (SK-MEL-5) cell lines. In the same paper [13  and caused apoptosis toward human T-leukemia cells of the Jurkat line. However, the application of 14 and 15 did not affect the morphology and the DNA intactness in mitogen-activated lymphocytes of the peripheral blood of healthy human donors.Finiuk et al.  reported4 and 15 , possessThe structural motifs of natural products always serve as an excellent source for drug-like molecules from the perspective of the design of anticancer agents ,28.16 and 17  and lung cancer cell line A549 (IC50 = 1.28 ± 0.98 μM). The cell cycle analysis disclosed that 25 showed significant G2/M phase arrest in MDA-MB-231 cells. Furthermore, hybrid 25 significantly inhibited tubulin polymerization, with an IC50 value of 3.54 ± 0.2 µM, and caused apoptosis-mediated cell death in MDA-MB-231 cells. In silico studies inferred that hybrid 25 binds at the colchicine binding site of the tubulin, with prominent binding affinity.The synthesis and evaluation of antiproliferative activity in A549 (lung cancer), MDA-MB-231 and BT-474 (breast cancer), HepG2 (liver cancer), and HCT-116 (colon cancer) cell lines using an MTT of coumarin-2–iminothiazolidin-4-one hybrids were reported by Sigalapalli et al. . Hybrid 25  showed eSteroids are important and physiologically significant natural compounds, opening up the possibility of structural tuning with the aim of achieving and developing valuable pharmacological properties including potent anticancer activity . Živković et al.  designed50 of the majority oscillated around 10 µM, with the IC50 of the cisplatin reference compound for these lines being 5.7 µM and 5.2 µM, respectively. Compared to the positive control, these compounds exhibited lower toxicity to normal cells. Hybrids 26 and 27 were found to be the most active . Furthermore, molecule 34 exhibited superior protection of normal HDF cells than 33. Additionally, the anticancer potency of the compounds was justified by using in silico studies. SAR analysis concluded by the authors suggests the importance of the presence of dichlorosubstituted arylamino moiety in position two of the 4-thiazolidinone core in enhancing potency.A series of 4-thiazolidinone-pyrazole hybrids connected via a ylidene linker were synthesized by Bhat et al. . An in v3 and 34  exhibite35 -3-(5-methylisoxazol-3-yl)thiazolidin-4-one, 42 , as a po42 exhibited excellent anticancer activity against MCF7 and displayed less cytotoxicity towards normal HEK293 cell lines. Hybrid molecule 42 inhibited the cell proliferation of HeLa by 58%, 87%, and 94% at 6.25 µM, 25 µM, and 50 µM concentrations, respectively. In terms of inhibition, 59% and 65% inhibition were observed at a 50 µM concentration on breast cancer (MCF7) and lung cancer (A549) cell lines, respectively, by compound 42. At lower concentrations, compound 42 has less effect on normal cell lines (HEK293). Additionally, a molecular docking study explored the binding mode and possible interactions between the synthesized compound and the ATP binding site of the EGFR kinase domain.The results of an in vitro cytotoxicity assessment employing the MTT assay as per the ATCC protocol, using three human cancer cell lines, HeLa , MCF-7 (breast cancer), A549 (human lung cancer), and HEK-293 , indicated that hybrid 43 (43 demonstrated inhibition activity against (GI50 < 10 μM) against all 59 human tumor cell lines, with average GI50/TGI/LC50 values of 2.57/57.27/94.71 μM, respectively. A series of 5-enamine–4-thiazolidinones with phenylalanine moiety in the molecules was designed and reported by Holota et al. . Among t43 , with a Preliminary SAR provided by the authors revealed that the presence of thiazole substituent is crucial for anticancer activity whereas a change in the hydrogen atom, aniline derivatives, or 1,2,4-triazole nucleus leads to the disappearance of the effect.44 and 45 were found to be the most active against cancer cell lines, with total mean GI50 values of 3.54 µM (44) and 10.96 µM (45), without causing toxicity to normal somatic HEK293 cells, with IC50 values of 28.99 µM (44) and 24.43 µM (45).Two series of merged 1,2,4-triazole–4-thiazolidinone hybrids with ylidene and enamine linkers in the molecules  were desAn SAR analysis performed by authors revealed that both the presence and position of a chlorine atom in the benzylidene area of molecules play a crucial role in the realization of the anticancer effect .46 and 47  and HepG-2 (liver cancer cell line) were IC50 values of 13.0 µM and 19.2 µM, respectively, for hybrid 46 and values of 12.4 µM and 16.2 µM, respectively, for 47.A series of 17 novel pyridine-thiazolidinone hybrids were synthesized, characterized, and evaluated as potential hCA inhibitors by Ansari et al. . Of all 6 and 47  had the 48 and 49  production, and modulated redox status.The molecular hybridization approach was applied for the synthesis of pyridine–4-thiazolidinone hybrids with aim of the synthesis of potential antiglioblastoma agents by Campos et al. . Among s50–52  displaye53 .The attempt to design potential antimitotic agents following the hybridization of pyridine, piperazine, and 4-thiazolidinone scaffolds was reported by Demirci et al. . The syn53  showed t54 , the IC50 value for the compound was 10.84 µM, indicating that compound 55 is 10-fold more selective against the colon cell line. SARs analysis performed by the authors revealed that the antiproliferative activity of phenethyl-thiazolidinone-indole hybrids is higher than that of benzyl-thiazolidinone-indole ones. Furthermore, it was observed that hybrids without a substituent at the nitrogen atom into indole core are more active than N-alkyl/aryl-substituted ones. In addition, the presence of the methoxy group at the phenyl ring of the side chain of hybrid compounds leads to higher activity than halogen substituents. Extended analyses on compound 55 revealed that the compound inhibited the cell cycle of HCT-15 cells in sub G1 and G2/M phases, additionally inhibited tubulin more strongly compared to the reference compound, induced intracellular ROS, and decreased the mitochondrial potential, causing cell death by apoptosis [Indole scaffolds are often used for the design and development of 4-thiazolidinone hybrids as anticancer agents. Sigalapalli et al.  reported55  showed tpoptosis .56  possessed moderate sensitivity to hybrid 57. Furthermore, compound 57 induced apoptosis in studied tumor cells via caspase-3-, PARP1-, and Bax-dependent mechanisms; however, it did not affect the G1/S transition in HepG2 cells. Additionally, hybrid 57 impaired nuclear DNA in HepG2, HCT116, and MCF-7 cells without intercalating this biomolecule, but much fewer DNA damage events were induced by 57 in normal Balb/c 3T3 fibroblasts compared with HepG2 carcinoma cells.A series of novel indole-azolidinone hybrids with 5-fluoro-3-formyl-1reaction . Among ttion [57  was foun®) [Isatin  is an en®)  was esta58  and MCF-10A , with IC50 values of 49.1 µM and 73.1 µM, respectively. In addition, hybrid 58 significantly induced the expression of the pro-apoptotic protein Bax, inhibited the expression of the anti-apoptotic protein Bcl-2, and increased the level of caspase-3 compared to the control sample.El-Naggar et al.  reported58  possesse59  activity. In addition, in silico and in vivo studies demonstrated that compound 59 has an excellent physicochemical and pharmacokinetic profile (which PK and docking can develop).Fouad et al.  develope59  was effe60  cell line was evaluated. The results obtained by the authors showed that the studied molecule 60 exhibits both cytotoxic and proapoptotic properties at a 10–100 µM concentration range. The activation of caspase-3 was caused by 60 and was accompanied by an LDH release and resazurin reduction. Additionally, increased DCF fluorescence in a wide range of concentrations after 6, 24, and 48 h of exposure was observed after treatment with 60, which suggests the stimulation of ROS production. The properties of 60 established by the authors make it a promising tool for further research as an anti-cancer agent.Szychowski et al.  reported60 . The imp61 , with the preservation of biochemical and histochemical structures being close to normal.Nafie et al.  studied 62  was the 63  that hybrid 63 arrested the cell cycle at the G2/M phase and possesses binding properties with N-acetyl transferase (hNAT-1) protein. The authors suggest that the identified quinolinone–4-thiazolidinone hybrid 63 could be considered as a new chemotype targeting hNAT-1 and could be used for hit/lead compound generation in anticancer drug design and discovery.A new series of quinolone–4-thiazolidinone hybrid molecules were reported by Kumar et al. . All the63  was founQi et al. ,65,66,6764,65,68) and toward HT-29 (human colon cancer cell line) (66–68) were identified.Structural modification of phenyl rings A and B as well as quinoline ring C was applied  in the mm-position in the phenyl ring attached to the 4-thiazolidinone nucleus at position two (69) was found to be the most active against the hepatic cell line, with an IC50 of 1.79 µg/mL. A series of hybrids with a quinazoline scaffold was synthesized by Samridhi Thakral et al.  Figure . The synp-position in the mentioned phenyl ring (70) leads to activity against breast cancer cell line MCF-7, with an IC50 of 1.94 µg/mL. The IC50 value for the standard drug in this study was found to be 0.09 µg/ml.The presence of the methoxy-group at the 50 values from 0.096 µM to 0.98 µM) on lung cancer (A549) cell lines, and four compounds showed a broad spectrum of cytotoxic activity on all the examined cancer cells in the range of 0.096–4.58 µM. Among them, compounds 71 and 72  of 0.096–0.32 µM and with low cytotoxicity in the HeK-293T line, with IC50 values of 6.76 and 6.65 µM respectively.Sharma et al.  reported1 and 72  were fou71 and 72 on A549 lung cancer cells revealed a remarkable inhibition of cell migration through the disruption of the F-actin assembly. Moreover, treatment with 71 and 72 led to the collapse of the mitochondrial membrane potential (DJm) and increased the levels of ROS in A549 cells. Reported in [In-depth studies of the impact of orted in  were theIqbal et al.  reported73–75  and extrinsic pathways, with the upregulation of anti-apoptotic signals and downregulation of pro-apoptotic signals, were the most often indicated. An important reported mechanism for antimitotic activity was an impact on the cell cycle with arrest following in different phases. Moreover, enzymes such as PIM-and tyrosine kinases as well as carbonic anhydrases were used as molecular targets for the design of novel 4-thiazolidinones with anticancer activity. Some reported hybrids were found to be promising tubuline inhibitors and inducers of ROS generation. The molecular hybridization strategy is a popular trend and an attractive research direction, providing endless source of significant opportunities in the design of new 4-thiazolidinone-bearing hybrid molecules with potential anticancer activity. A combination of potential pharmacophores in one molecule is one of the possible approaches used to achieve multi-target and polypharmacological effects for small molecules and is especially essential in the case of antimitotic agents. The hybridization of scaffolds, the hybrid-pharmacophore approach, and analogue-based drug design are key tools that are widely used by synthetic and medicinal chemists in this field. The hybridization of the 4-thiazolidinone core with early approved drugs, natural compounds, and privileged heterocyclic scaffolds is the most frequently used method in the development of novel molecules with antimitotic effects. The application of a molecular hybridization methodology allowed for identification of the new “hit” and “lead” hybrid compounds which target valid and important targets in carcinogenesis. The mentioned strategies and methodologies will remain relevant to studies over the next decade, and new wave/direction in the design of potential antimitotic agents using the hybridization of 4-thiazolidione scaffolds with biomolecules such as monoclonal antibodies, etc., can be expected. The data presented in this review contribute to the SAR profile of 4-thiazolidinone-based hybrid molecules for further exploration in the design, improvement, and optimization of new molecules with antitumor activity.


The plasma membrane separates the interior of the cells from the extracellular fluid and protects the cell from disruptive external factors. Therefore, the self-repairing capability of the membrane is crucial for cells to maintain homeostasis and survive in a hostile environment. Here, we found that micron-sized membrane pores induced by cylindrical atomic force microscope probe puncture resealed significantly (~1.3–1.5 times) faster in drug-resistant non-small cell lung cancer (NSCLC) cell lines than in their drug-sensitive counterparts. Interestingly, we found that such enhanced membrane repairing ability was due to the overexpression of annexin in drug-resistant NSCLC cells. In addition, a further ~50% reduction in membrane resealing time  was observed through the epithelial-mesenchymal-transition, highlighting the superior viability and potential of highly aggressive tumor cells using membrane resealing as an indicator for assessing the drug-resistivity and pathological state of cancer. In addition to serving as the physical barrier separating cytoplasm from outside, and protecting the cell from disruptive extracellular factors, the plasma membrane also allows a cell to achieve signal and mass exchange with the surrounding microenvironment  and maintain homeostasis is critical for the cell’s survival. For example, it is widely reported that cells must actively pump out calcium ions to keep their internal concentration low, a condition that is critical for the proper functioning of many proteins and signaling pathways . HoweverMicroscopically, it is well-documented that the calcium influx through the ruptured membrane could trigger a cascade of events to help its repairing. In particular, annexin family proteins  were used in this study. The HCC4006 cell line was cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS), and ETCC016 cells were cultured in DMEM supplemented with 10% FBS and 1% PS. Both cell lines were maintained in a 37 °C humidified incubator containing 5% COBoth HCC4006 and ETCC016 cell lines were treated with 5 μM erlotinib for over 10 passages to gain resistance to the drug. We named the resistant phenotype of these cell lines as HCC4006R and ETCC016R, respectively, whereas the parental control groups  were treated with the same amount of dimethyl sulfoxide (DMSO), instead of erlotinib.To evaluate the IC50 value of erlotinib in both drug-sensitive and drug-resistant cell lines, MTS assay CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit  was performed. Briefly, 0.0001 µM, 0.001 µM, 0.01 µM, 0.1 µM, 1 µM, 10 µM, 20 µM, 50 µM, 100 µM, 200 µM erlotinib and control (DMSO) were added to both cell lines. After 72 h, the supernatant was discarded and replaced with MTS reagent solution (20 µL/well for 96-wellplate), and cells were then incubated for another 4 h. The absorbance of 490 nm wavelength was read by a plate-reader to measure the viability of cells.EMT in both HCC4006 and ETCC016 cell lines was activated through TGF-β receptor signaling pathways. Specifically, 4 ng/mL TGF-beta was added to the growth medium 36 h before experiment . The occCells were digested and transferred into glass-bottom dish  precoated with poly-L-lysine , tested after 30 min incubation to ensure cells would adhere strongly to the dish. Membrane puncturing experiment was conducted using atomic force microscopy (AFM). Specifically, cylindrical AFM probes (with a diameter of 2 µm) were fabricated by focused ion beam (FEI Quanta 200 3D). After that, said probe was pushed into the cell to induce membrane damage (similar to that achieved by using laser or electric pulses ). The recP) and external (eP) pressures were balanced by the membrane tension γ m via the Laplace’s law asR represents the cell radius. Since the indentation depth is small, the contact area A and indentation depth δ can be related to each other as:The membrane tension of cells was estimated by indenting the cell membrane with a cylindric AFM, a method that has been used in previous studies ,20. BefoF applied on membrane through the cross-section area A of the indenter must equal to that induced by the pressure difference across the membrane, i.e.,On the other hand, the force Combining the above equations, the membrane tension can be estimated as:Cells were treated with cold methanol for 10 min to make their membrane permeable to dyes (without destroying embedded membrane proteins), followed by another 10 min treatment of PBS with 0.25% Tween 20. After being washed with PBS for 3 times, the samples were immersed in 5% BSA blocking solution for incubating 30 min at room temperature. Next, primary monoclonal antibody annexin A2  diluted in 2% BSA PBS and added. After overnight incubation at 4 °C and washing with PBS 3 times, a secondary antibody was added and the samples were incubated at room temperature for 1 hr. DAPI  was added into the medium 30 min before observation.The fluorescence images were obtained using Nikon Eclipse Ts2R-FL fluorescence microscope with fixed exposure time for each sample.To evaluate the expression of EMT markers, real-time quantitative PCR (RT-qPCR) was employed to detect the expression levels of E-cadherin, N-cadherin and vimentin. To further determine the role of proteins in relation to membrane repairing capability, annexin A2, A5, A6, A7 were also tested. GAPDH was determined as the reference gene. The primers were purchased from Lifeact Technology . TRIzol Reagent  was used to lyse the cell to extract mRNA, following the manufacturer’s instructions. RT-qPCR was performed with a QuantiNova SYBR Green PCR Kit . Specifically, the cycling program was adopted as: (i) denaturation at 95 °C for 15 s, (ii) annealing at 60 °C for 30 s, and (iii) repeating these two steps for 45 cycles .Using an atomic force microscope (AFM) mounted with a 2 μm cylindrical probe refer to a, membraWith the method for introducing membrane pores with precisely controlled size at hand, we can then examine how the self-repairing capability of cancer cells is influenced by their pathological status. Given its important clinical implications, we first investigated how the drug resistance developed in non-small cell lung cancer cells affect their membrane resealing ability. Specifically, drug-resistant ETCC016 and HCC4006 cells were developed by culturing these two cell lines with 5 μM erlotinib for more than 10 passages. The resistance to erlotinib of these cell lines was evaluated/verified by the MTS assay shown in As pointed out earlier, membrane tensions in these drug-resistant and drug-sensitive cells were found to be more or less the same, therefore we look for other factors that could give arise to the different membrane resealing response observed here. In particular, we examined the expression of annexin in these cell lines, given the well-known role of such proteins in facilitating membrane remodeling. As shown in Another way for tumor cells to acquire drug-resistance is the EMT, where epithelial-like cells lose their apical-basal polarity and adhesion with each other, and eventually become highly migratory. Specifically, the reduced drug-induced apoptosis and elevated drug efflux pumping ,25 in tuWe proceeded by adding 4 ng/mL TGF-β, a common EMT inducer, into the culture medium of cells. Interestingly, as shown in It must be pointed out that, among all cell lines tested, HCC4006R underwent the largest increase in the annexin expression level (after TGF-β treatment) coupled with the biggest decrease of membrane resealing time (from ~23 to ~13 s) which again supports the notation that EMT enhances the membrane repairing capability of cells. Similar to the previous drug-sensitive and drug-resistant case, we then examined how TGF-β treatment influences annexin expression in NSCLC cells. As expected, a much bigger increase in the expression level of annexin A2, A5, A6 and A7 was observed in drug-resistant HCC4006R cells, in comparison to the HCC4006M cell line a. On theThis study showed that micron-sized membrane pores (induced by AFM puncturing) in NSCLC cells could fully reseal in tens of seconds. Interestingly, although membrane tension remained more or less the same across cancer cell lines, erlotinib-resistant tumor cells were found to reseal their membranes much faster than the corresponding drug-sensitive cell lines. We further demonstrated that such enhanced membrane repairing capability was due to the overexpression of annexin (a protein family known to play significant roles in the remodeling and reorganization of lipid membranes) in drug-resistant NSCLC cells. Interestingly, EMT could further reduce the resealing time of the membrane. These findings are consistent with previous studies reporting that annexin facilitates membrane repairing by inducing curvature and folding of lipid bilayer near the pore edge , and knoIt must be pointed out that clinically, overexpression of annexin A2 in the tumor was considered a predictor of adverse outcomes for patients . Recent Finally, given that membrane puncturing and resealing tests can be conducted in a few minutes, it is conceivable that we might be able to use membrane resealing time as a marker for rapid drug-resistance identification and/or classification of cancer. Indeed, cell stiffness level has been successfully applied to distinguish tumor cells from their normal counterparts . Finally


Carcinomas are characterized by a widespread upregulation of intercellular junctions that create a barrier to immune response and drug therapy. Desmoglein 2 (DSG2) represents such a junction protein and serves as one adenovirus receptor. Importantly, the interaction between human adenovirus type 3 (Ad3) and DSG2 leads to the shedding of the binding domain followed by a decrease in the junction protein expression and transient tight junction opening. Junction opener 4 (JO-4), a small recombinant protein derived from the Ad3 fiber knob, was previously developed with a higher affinity to DSG2. JO-4 protein has been proven to enhance the effects of antibody therapy and chemotherapy and is now considered for clinical trials. However, the effect of the JO4 mutation in the context of a virus remains insufficiently studied. Therefore, we introduced the JO4 mutation to various adenoviral vectors to explore their infection properties. In the current experimental settings and investigated cell lines, the JO4-containing vectors showed no enhanced transduction compared with their parental vectors in DSG2-high cell lines. Moreover, in DSG2-low cell lines, the JO4 vectors presented a rather weakened effect. Interestingly, DSG2-negative cell line MIA PaCa-2 even showed resistance to JO4 vector infection, possibly due to the negative effect of JO4 mutation on the usage of another Ad3 receptor: CD46. Together, our observations suggest that the JO4 vectors may have an advantage to prevent CD46-mediated sequestration, thereby achieving DSG2-specific transduction. Carcinomas, malignancies of epithelial tissue, represent the majority of all human cancer cases . The key6 PtDd per infectious virus), and it has been hypothesized that PtDd contributes to virus escape and spread [Adenoviruses have evolved mechanisms to breach epithelial barriers . For exad spread .To address the issue of the reduced carcinoma penetration rates of systemically applied oncotherapy, the high-affinity junction opener 4 (JO-4) was developed by mutagenic libraries . JO-4 prMacaca fascicularis [The binding of JO-4 protein to DSG2 leads to the shedding of DSG2 followed by a decrease in junction protein expression and transient tight junction opening. JO-4 protein’s synergy with antibody and chemotherapy was proven in efficacy studies with the chemotherapy drug Doxil  and ovarian cancer in a mouse xenograft model . Furthericularis . Currenticularis ,27. Receicularis . This HDPreviously, other high-affinity fiber knobs from Ad35 were explored to improve the in vitro and in vivo infection properties . For theOur study aimed at producing high-DSG2-affinity viral vectors to improve cell uptake and tumor penetration depth. To this end, we constructed three novel vectors containing the JO4 mutation and investigated them in a panel of tumor cell lines with different DSG2 expression levels to assess these vectors for their infectivity, transgene expression ability and oncolytic effects in direct comparison with their parental viruses. We found that the high-affinity JO4 mutation did not result in correspondingly higher transduction rates of individual viruses. However, we observed that the DSG2 level played a crucial role in the JO4-containing viruses’ activity. In the high-DSG2-expressing cell lines Hs578T and Hela, the JO4-containing viruses displayed comparable transduction levels with their individual parental viruses. In comparison, in the DSG2-negative cell line MIA PaCa-2, the JO4 vectors showed significantly weaker effects than their parental vectors. We further analyzed the effect of JO4 on the usage of CD46 and CAR, another two major adenovirus receptors. CD46 is proven to be a binding and entry receptor for Ad3, especially in the absence of DSG2 ,34. HereThe JO4 mutation was introduced into the adenoviral genome containing plasmids as previously described ,36,37. ITo rescue these vectors, the plasmid backbone was removed with pre-inserted restriction enzymes; the released adenovirus genome containing the aimed for modification was transfected into HEK293 cells with calcium–phosphate transfection reagents. After serial passaging to amplify the vectors to large scale, the vectors were purified in two rounds of cesium chloride (CsCl) gradient, as previously described . The ade2.Cell lines Hela, HEK293, Hs 578T, HNC, Huh 7, MDA-MB-231, CHO, Caco-2, MIA PaCa-2 and A549 were cultured in high-glucose Dulbecco’s modified Eagle’s medium . CHO cells were further supplemented with 1% non-essential amino acid solution . T84 was supplied with half DMEM, half Ham’s F12 (PAN-Biotech). Jurkat and K562 cells were cultured in RPMI 1640 (PAN-Biotech). All the above media were supplemented with 100 units per mL penicillin and 100 µg per mL streptomycin (PAN-Biotech). The Caco-2 medium was supplemented with 20% FBS, while all other media contained 10% FBS from the same supplier (PAN-Biotech). All cells were maintained in a humidified atmosphere at 37 °C and 5% COFor the transwell culture experiments, T84 cells were seeded into collagen-coated transwells . For this purpose, cells were counted, and 80,000 cells were seeded per well. Cells were cultured in T84 medium see  from apiTo form spheroids, approximately 1000 cells per well were seeded of different carcinoma cell lines into U-bottom 96-well plates  in their respective media. Brightfield and fluorescence images were taken every 12 h using a Satorius Incucyte SX5 live cell imaging system. Numeric evaluation was performed with the Incucyte spheroid software .5 cells were seeded in 24-well plates. After achieving a complete confluence, cells were infected at various viral particles per cell (vp/c) and incubated overnight. After 24 h, cells were washed once with Dulbecco’s Phosphate Buffered Saline (DPBS) and detached with trypsin-EDTA. Cells were resuspended in DMEM containing 10% FBS, followed by a centrifugation step , and washed in DPBS before fixation in 2% paraformaldehyde (PFA). Fluorescence profiles were obtained by analyzing at least 10,000 viable cells on a CytoFlex flow cytometer . A background signal was obtained by analyzing the negative control consisting of uninfected cells. The percentage of GFP-expressing cells was determined by selecting the region of fluorescence above the background of auto-fluorescence from uninfected cells.To detect the transduction efficiencies in different tumor cell lines, cells were analyzed with flow cytometry. First, 1 × 105 cells were washed with PBS supplemented with 1% BSA, centrifuged  and resuspended in 100 μL PBS/BSA and 1 μL PE-conjugated rabbit anti-hCAR antibody . Following an incubation step at room temperature for 1 h, cells were washed again with PBS/BSA to remove unbound antibodies and resuspended in 100 μL PBS for flow cytometry (Beckman Coulter CytoFlex flow cytometer). The PE-conjugated mouse anti-human CD46 antibody  was used to detect the surface expression of CD46 on different cell lines. For the DSG2 detection, Alexa Fluor 488-conjugated mouse anti-human Desmoglein 2 antibody was used .To detect CAR (coxsackievirus and adenovirus receptor) expression on the cell surfaces of different tumor cell lines using flow cytometry, 1 × 10The TEER of the T84 cellular barrier was measured in ohm to evaluate the barrier integrity. The transwell culture, which consisted of a multiple layer of cells cultured on a semipermeable filter insert, produced a partition for the inner (or upper) and outer (or lower) chambers. For the electrical measurements, Millicell ERS-2 Voltohmmeter  was applied. Two electrodes, one placed in the inner and one in the outer chamber, were used to measure the resistance of the cellular multilayers.The histological analysis of the T84-transwell cultures was performed to visually evaluate cell differentiation and integrity. Briefly, the 3D cultures were fixed in 4.5% formaldehyde/PBS. After 24 h of fixation, the round membrane was cut out from the transwell using a sterile and sharp scalpel and padded on blue tissue foam . This was subsequently soaked in tap water for 2 h. After dehydration in alcohol, clearing in xylene and embedding in paraffin, the blocks were cut to a thickness of 5 µm with a rotary microtome . Then, the slides were incubated overnight at 37 °C. The blocks were then de-paraffinized at high temperatures, followed by xylene treatment and rehydration with ethanol and washing with deionized water. Subsequently, hematoxylin and eosin staining were performed. The sections were examined under a microscope.To quantify the cell entry efficiency, a defined number of adenovirus particles (vp) was used to infect pre-seeded cells in 24-well plates, which were then incubated for different time periods. Cell monolayers were digested and flushed off with trypsin, followed by intensive washing with PBS. Genomic DNA was extracted using Monarch Genomic DNA Purification Kit . To monitor virus genome uptake efficiency, quantitative polymerase chain reaction (qPCR) analysis was performed.To monitor virus genome uptake efficiency, duplex-qPCR was performed with primers and a probe, detecting the Ad3 fiber knob region  to quantOncolytic assays were performed in 48-well plates. A 3-fold dilution series of individual adenoviruses was freshly prepared to infect pre-seeded cancer cells. The development of any cytopathic effect (CPE) was checked daily until at least one of the viruses on one plate at the lowest dosage showed CPE. The experiment was maintained for a maximum of 14 days. The cells were first fixed with 3.7% formaldehyde, and then the maintained adherent cells were detected by staining the attached cells with 0.5% crystal violet dye. After several wash steps, the stained plates were dried and photographed.Plaque assays were performed following a previously published protocol . In deta9 vector particles was added to SDS-loading buffer, heated for 5 min at 70 °C and loaded on an 8% separation/5% stacking SDS-gel. For Ad3 and Ad3–JO4, a 4–15% gradient gel Mini-PROTEAN TGX  was used. Both gels ran at 80 V. The separated vector proteins were stained silver as previously described [An amount of 1 × 10escribed ,41.Statistical analyses were conducted with GraphPad Prism 8 . We used the nonparametric Mann–Whitney U test to identify significant differences between the JO4 vectors and their individual parental vectors .The aim of this work was to transfer the previously identified junction opener protein 4 (JO-4) into a viral vector setting. We first generated viral vectors with the JO4 mutation and then explored the effect of different JO4-containing adenoviral vectors in their infectivity, transgene expression ability and oncolytic effect in direct comparison with their individual parental vectors. To test the potential therapeutic benefits of the JO4 variant, we explored different cancer cell lines in two-dimensional (2D) and 3D culture regarding transgene expression level and virus cellular entry. Moreover, to investigate the correlation between virus infection and receptor expression, cell lines with different major adenovirus receptor expression levels were included in this study. Furthermore, these vectors were evaluated in CAR and CD46 knockout A549 cells as well as in stable CD46- and CAR-expressing CHO cells.To investigate the affinity-enhancing JO4 mutation in the context of a virus, we constructed three new vectors containing the JO4 mutation (JO4 vectors) A–C. The To confirm that all JO4 vectors are properly expressing fiber protein, the virus particles were lysated with SDS containing buffer and loaded on silver gel. As shown in To explore the effect of JO4 on virus infectivity, the JO4-containing vectors and their parental vectors were investigated in a panel of tumor cell lines. A previous study of CD46 high-affinity Ad35 fiber knob (Ad35++) revealed that the CD46 density played an essential role in the cell line transduction . TherefoFirst, we performed the experiments in monolayer cell culture. Since colon adenocarcinoma-derived Caco-2 and T84 cell lines are frequently used as in vitro models of functional epithelial barriers, we chose these two as models for analyzing the high-affinity DSG2 vectors. The two cell lines showed similar major adenovirus receptor levels (CD46 and CAR) and around 75% DSG2 expression . Here weAs many cancers with hematopoietic cell origin show strongly upregulated DSG2 expression and present a potential target for oncolytic viral therapy , we alsoThe ability to spread in a tumor is an important feature for the application of oncolytic viruses in solid tumors. Here we investigated the JO4-containing vectors in monolayer cell culture regarding plaque forming and cell lysis .We first assessed the two replication-competent viruses Ad3 and Ad3-JO4 in A549 cells and in the CD46 knockout A549 cell line (A549-CD46-KO) by applying an agarose overlay to evaluate the virus’s spreading ability. No difference was observed between the two viruses regarding plaque size or number A. It wasFurthermore, we investigated the Ad3-JO4 vector in spheroid culture, which has been used in cancer research as an intermediate model between in vitro cancer cell line cultures and in vivo xenograft tumors. We used a panel of human carcinoma cell lines that were able to grow as dense spheroids and showed high DSG2 expression (over 80% of cells expressed DSG2) including MDA-MB-231 (breast), Hela (cervix), Huh 7 (liver) and HNC (head and neck) cells A–D. An aSince the natural target of Ad3 is polarized epithelial tissue, we then studied the spread of Ad3 and Ad3-JO4 in a colon cancer T84 cell transwell culture model. T84 cells were cultured on semipermeable filter inserts in order to form a multilayer structure with intercellular junctions for four weeks. Amounts of 400 vp/c of Ad3 and Ad3-JO4 were added to the apical chamber. Trans-epithelial electrical resistance (TEER) was measured at the indicated time points E. There Since we observed a correlation of DSG2 expression level and JO4 vector behavior, we investigated JO4 vectors’ usage of CD46 and CAR. As the CHO-K1 wild-type cell line expresses none of the three primary adenovirus receptors, CD46, CAR or DSG2, and allows neither Ad3 nor Ad3-JO4 to transduce, it is a suitable model for studying the interactions of these two vectors with CD46 or CAR exclusively, using stable CD46- or CAR-expressing CHO cell lines A. The FAFurthermore, we evaluated all three JO4 vectors in comparison with their parental vectors in wild-type A549 cell line, CD46 or CAR knockout and CD46 and CAR double- knockout A549 cell lines B–E. Ad5 Virus attachment and cell entry are the first steps of infection, and the latter is also used to define the infectious titer or transducing units. Here we quantified the virus genome inside the cells at certain time points postinfection in qPCR . In HEK Viruses can interact not only with well-displayed receptors at the apical side of epithelial cells but also with poorly accessible receptors hidden in cell to cell junctions . The molNotwithstanding its promising application as a recombinant protein, definitive proof of the function of JO4 in the viral life cycle is elusive. Therefore, we generated three adenoviral vectors with the JO4 mutation in the fiber knob. The first two groups are replication-defective chimeric vectors based on E1-deleted Ad5, with either the whole fiber region from Ad3 (F3) or only the fiber knob from Ad3 (K3). The third pair are replication-competent Ad3 vectors. The transduction was first evaluated in a panel of tumor cell lines with DSG2 levels ranging from zero to one hundred percent receptor-positive cells . SurprisIn two-dimensional (2D) submerged culture, we did not observe any difference with plaque-forming assays but saw three- to ten-fold less efficiency of Ad3-JO4 compared with Ad3 in oncolytic assays, suggesting the reduced virus transduction of these tumor cell lines . Since tBased on the distinguishing features of JO4 viruses in cancer cell lines with different DSG2 levels, we hypothesized that not only DSG2 binding but also interaction with other molecules or the lack thereof played a crucial role in the life cycle of JO4 viruses. In addition to DSG2, CD46 has proven to be a minor receptor for Ad3 infection, and CAR was also hypothesized as such ,34,47. TWe suspect that the valine in position 239 of the fiber knob amino acid sequence serves as a region crucial for Ad3 fiber knob binding to CD46. Moreover, human A549 cells and individual CD46 and CAR knockout cell lines were included. However, here the receptor knockout did not induce a difference in virus transduction between with and without JO4 B–E. We sA receptor density effect was previously observed in high-affinity Ad35 fiber knob containing viruses (Ad5/35+ and Ad5/35++) . No signCurrently, Ad5-based chimeric vectors with fiber knobs from Ad3 and pure Ad3-based vectors represent promising alternatives to traditional Ad5 vectors in terms of receptor usage and preexisting immunity ,55,56; iIn sharp contrast to the efficacy observed with the single JO-4 protein, the JO4 mutation resulted in no enhanced efficiency in the context of a virus, probably due to the negative influence of this mutation on CD46 interaction. Therefore, JO4-containing vectors should be considered in applications to compete with the CD46-mediated sequestration of vector particles in directing DSG2-specific transduction.


Lavandula angustifolia is a flowering plant that possesses anti-inflammatory and anti-depressive medicinal properties. Cancer is a deadly disorder affecting millions of people globally. It affects several human organs, including liver, stomach, and lungs. Several researchers are doing efforts to eliminate the disease around the globe. In this study, Chang and Huh-7 liver cell lines were utilized as human normal hepatocyte model and innovation to mimic the liver environment. Cytotoxicity of L. angustifolia seed extracts was investigated at two different concentrations (50% and 100%) against Chang and Huh-7 liver cell lines by colorimetric assay which is used to assess cell metabolic activities. The Chang and Huh-7 liver cell lines were treated with L. angustifolia seeds extracts (50% and 100%) and incubated for 24 and 48 hours under standard conditions . The 3--2, 5-diphenyltetrazolium bromide assay was employed to quantify cell survival. Seed extracts of L. angustifolia exerted varied cytotoxic effects depending on the concentration and treatment duration. The results indicated that L. angustifolia seed extracts with 100% concentration exhibited the highest cytotoxicity against Huh-7 and Chang liver cell lines. In conclusion, L. angustifolia seed extracts exhibited cytotoxic activity which can be enhanced based on the concentration and treatment duration. The findings of the current study are critical for the development of novel herbal-based therapies for fatal disorders such as liver cancer. However, more investigations are required to reveal cytotoxicity mechanisms of L. angustifolia seed extracts.Flowering plants are valuable in numerous ways, including food/feed supply for living organisms, fuel production, and medicinal uses. Several plant extracts/products are used to treat variety of serious ailments in human and animals.  Several diseases pose significant threats to human life. Although medical science has witnessed significant progress in the twenty-first century, there are still several diseases that are incurable. Therefore, cures for these diseases must be investigated. Cancer is a fatal health issue that affects people all over the world. It can arise in almost any tissue/organ of the body when anomalous cells grow uncontrollably, migrate to other organs through metastasizing process and cause death . Cancer in vitro model. The HuH-7 cell line was developed in 1982 from a 57-year-old Japanese male liver tumor patient’s hepatocyte cell line. It was then further refined and now being used in vitro to produce hepatitis C virus (HCV) medicines. The unique characters of HuH-7 cell line reveal that it is made up of epithelial-like, tumorigenic cells that stick to the surface of flasks or plates and proliferate as 2D monolayers. According to detailed literature review, this is the first study investigating the impact of lavender seed extracts on liver cancer cells.Different types of plants have been used in herbalism in various parts of the world in all civilizations at local level. The plant-based products are being suggested by professionals for the treatment of a variety of disorders. Numerous people around the world use herbal medications for a variety of medical needs. Plant species are thought to be a rich source of compounds that can be utilized for making active pharmaceutical or manufactured medications. Plant-based medicines are usually free of adverse effects, which makes them more popular among the public. Plant extracts serve various purposes, including natural coloring, crop pest management, food, cosmetics, and beverage etc. Natural plant species have been used not only as sources of food, but also as remedies for many diseases in the ancient era. Different reports suggested that there are unidentified properties in different parts of plants that can be used in the treatment of certain diseases . The curLavandula angustifolia Mill (LV) ] belongs to the mint family that is native to Mediterranean, Middle Eastern Asian countries, Arabian Peninsula, and Russia, also growing in Europe, United States, and Australia  methanol extracts at concentrations from 20 to 2400 μg/ml 24 hours after incubation are shown in Stereological investigation of Huh-7 liver cells’ viability exposed with L. angustifolia methanol extracts. Cell viability was not affected by LV50%, and LV100% methanol extracts-treated Chang liver cells at concentrations <200μg/ml, whereas significantly decreased by the concentrations >200μg/ml after 24 h incubation compared with control cells.Researchers around the world are looking at the use of plant-derived medicines as a healthcare supplement . Herbal L. angustifolia seed extract by indicating its cytotoxic property against Hun7 cells, human hepatocellular carcinoma cells, and Chang liver cells, explaining the cell death mechanism in vitro.Hepatocellular carcinoma (HCC), a type of malignant tumors, is causing high mortality of cancer-related death worldwide. Compared to other forms of cancers, HCC arises from several stages and multifactorial progression. Different risk factors drive the progression of HCC malignancy, and treatments are not effective once the disease is initiated , 27. TheL. angustifolia seed extract has cytotoxic-inducing effects in Huh7 and Chang liver cells in a duration and concentration-dependent mode. The cytotoxic property could be due presence of different pharmacological active principles in LV50% and LV100% seed extract. Based on the result, we found that 100% methanol extract of L. angustifolia seed extract increased cytotoxicity at 48 h compared to other time. A similar pattern of results we observed that Tridham and its active principles showed cytotoxic property in the human cancer cell lines [L. angustifolia grown in Brazil against GM07492 human fibroblasts cells [L. angustifolia exhibited a promising cytotoxicity against HeLa and MCF-7 cancer cell lines. Another recent work investigated the antiproliferative activity of L. angustifolia essential oils that grown in Tuscany  showed a notable effect against SHSY5Y neuroblastoma cell line [L. angustifolia seed extract induces active growth inhibition in hepatoma cells. Presence of several phytochemicals such as carvacrol, spathulenol, p-cymene-8-ol, caryophyllene oxide, and terpinolene present in L. angustifolia seed extract are responsible for the obtained results. Salleem et al. [We found that ll lines . Our fints cells . In addits cells , who fouell line , and them et al.  reportedL. angustifolia seed extract may provide the possibility of cancer prevention and therapy, which is evidenced by the inhibition of cytotoxicity in cancer cell lines. Among the two tested doses, 100% methanol extract exhibited promising cytotoxic properties. Further, additional investigations are warranted to delineate proper molecular mechanisms involved in the anticancer activity.In conclusion,


Appropriate sample preparation is critical for the analysis of cell cultures with mass spectrometry. It is important not only to maintain clear and consistent morphological features, but also the local chemical composition of the sample. In this proof-of-concept study, we evaluated two different sample preparation procedures for proteomic analysis using imaging mass spectrometry (IMS) of formalin-fixed multiple myeloma (MM)-cultured cells. Cytospin preparation resulted in more peaks with a signal-to-noise ratio > 3 as compared to formalin fixation and paraffin embedding. Overall, IMS technology holds the potential to stratify different cell lines to address the identification of differentially expressed proteins. We propose this approach as an additional feasible method for proteomic investigation of MM cell lines, and potentially applicable to other tumor types.Sample processing of formalin-fixed specimens constitutes a major challenge in molecular profiling efforts. Pre-analytical factors such as fixative temperature, dehydration, and embedding media affect downstream analysis, generating data dependent on technical processing rather than disease state. In this study, we investigated two different sample processing methods, including the use of the cytospin sample preparation and automated sample processing apparatuses for proteomic analysis of multiple myeloma (MM) cell lines using imaging mass spectrometry (IMS). In addition, two sample-embedding instruments using different reagents and processing times were considered. Three MM cell lines fixed in 4% paraformaldehyde were either directly centrifuged onto glass slides using cytospin preparation techniques or processed to create paraffin-embedded specimens with an automatic tissue processor, and further cut onto glass slides for IMS analysis. The number of peaks obtained from paraffin-embedded samples was comparable between the two different sample processing instruments. Interestingly, spectra profiles showed enhanced ion yield in cytospin compared to paraffin-embedded samples along with high reproducibility compared to the sample replicate. Mass spectrometry (MS)-based proteomics is an emerging approach to generate protein signatures from clinical samples for biomarker discovery ,2,3,4. IIn order to use IMS-based proteomics for the analysis of cell cultures in routine research and possibly in the clinical setting, the variability associated with the sample preparation steps that precede IMS analysis must be considered. We used multiple myeloma (MM) as a model system to establish an IMS experimental procedure for cultured cells.Multiple myeloma (MM) is a B cell malignancy of clonal plasma cells (PCs) accumulating in the bone marrow (BM), characterized by an intricate genomic and epigenomic landscape. Despite the remarkable therapeutic achievements that have led to an increased extent and frequency of response with unprecedented outcome improvement, MM patients may eventually evolve towards a drug-resistant phase ,23,24. MS-based methodologies have proven to be a valuable diagnostic tool for minimal residual disease monitoring in plasma cell dyscrasias, including MM . AdditioIn order to further assess the clinical utility of MALDI MS, we optimized the sample preparation workflow for the proteomic investigation of MM cell cultures by comparing two different sample processing methods, including the use of the cytospin sample preparation, formalin fixation and paraffin embedding, and automated sample processing apparatuses. Herein, we present an optimized methodological approach, comparing two sample-embedding instruments using different reagents and with different processing times. We evaluated the peptide molecular extraction and the quality of mass spectral data obtained from fixed cell specimens with both procedures.MM cell lines were cultured in RPMI-1640 medium  supplemented with 10% FBS and 1% P/S (Gibco) . The AMOFor each cell line, the supernatant liquid was decanted in a separate tube and cell pellets were removed with a disposable Pasteur pipette and placed into a Tissue-Tek Paraform biopsy centralized cassette . One third of the cell pellets was resuspended in 70% ethanol and stored at 4 °C until further use. The remaining cell pellets were then introduced in the automated tissue-processing machine , where they were transferred from container to container to be processed. The pellet of the cell line “AMO BZB (8425)” was processed with a different automatic machine  employing a similar solvent composition but a longer reagent incubation time. To create paraffin-embedded specimens, the tissue processors employ raised temperatures, a low-wattage microwave assistance (60 watts), and a vacuum infiltration technique to enhance processing and reduce the processing time from 8 h on a conventional processing platform to only 2.5 h when using Sakura Tissue-TEK-VIP, and to about 1 h for the Sakura Tissue-TEK-Xpress. FFPE cell sections (3 µm) were mounted onto indium-tin oxide-coated (ITO) glass slides  and dried at 37 °C overnight. Deparaffination occurred by heating the slides at 80 °C for 15 min, followed by incubation in xylene (2 × 5 min). Slides were dehydrated in 100% isopropanol, followed by graded ethanol  and deionized water washes, for 3 min each. Antigen retrieval was performed by heating the slides at 95 °C for 20 min in 10 mM of Tris buffer pH 9 using a decloaking chamber , followed by enzymatic retrieval using trypsin. Trypsin solution (0.025 µg/µL in 20 mM of ammonium bicarbonate) was applied on the samples with an automatic reagent sprayer  in 16 passes at 30 °C, with a 0.015 mL/min flow rate and a 750 mm/min velocity. Samples were then placed in a digestion chamber, prepared with saturated potassium sulphate solution, and incubated at 50 °C for 2 h.The resuspended cell pellets were mixed by pipetting, and 150 µL of the cell suspensions was cytocentrifuged onto ITO-coated glass slides  in a cytospin  at 1200 rpm for 10 min. Slides were then incubated in 10 mM of Tris buffer pH 9, together with the dewaxed sections described above for the antigen retrieval process, and air-dried trypsin solution (0.025 µg/µL) deposition onto cytospin slides, and the digestion process occurred using the same method as described for the FFPE cell sections .Digested samples were sprayed with a matrix solution  using the same sprayer apparatus in 4 passes at 75 °C, with a 0.120 mL/min flow rate and a 1200 mm/min velocity. MALDI MS data were acquired using a rapifleX MALDI Tissue-typer mass spectrometer  in positive-ion reflector mode in a mass range of 500–3200. Each mass spectrum was obtained by averaging signals from 200 laser pulses. External calibration was performed using the peptide calibration standard  . All mass spectra were baseline-subtracted during acquisition with the TopHat algorithm. Following mass spectra acquisition, cells were stained with hematoxylin and eosin.Spectral data were pre-processed using FlexAnalysis 4.2, ScilS Lab software, version 2022b Pro , and R-statistical software, version 3.5.3 and 4.0.3, for total ion count (TIC) normalization, mass alignment, peak counting, and feature detection. Statistical analysis using Wilcoxon/Kruskal–Wallis tests and receiver operating characteristic curve (ROC) analysis was performed to measure the discrimination quality of the ion peptide features between the cell lines.Examination of the stained cells by light microscopy revealed a nice balance of nuclear and cytoplasmic stains in both types of cell sample processing methods, however cytospin-stained cells showed better cellular architecture and resolution of nucleoli .m/z range 500–3000). The median total peak count and signal-to-noise ratios (S/N > 3) were used as quality parameters to compare all experiments. For each sample procedure, the overall amount of the peaks detected in the same cell line and for each individual replicate was similar . Nonetheless, the highest peak counts and lowest variance were achieved in all cytospin samples  and FFPE (m/z with log2 intensity cytospin/FFPE < 0 = 145) was obtained, indicating that the higher number of peaks for cytospin is not related to a higher general peptide ion intensity (m/z < 800) were likely to have a higher intensity in cytospin than in FFPE, and conversely peaks in the high mass range (m/z > 800) had a higher intensity in FFPE than in cytospin (m/z < 800). In order to improve data quality and to prevent characteristic discrepancies between data collected from different samples, it Is recommended to extract spectra from regions of interest sufficiently far from areas with few cells. This would reduce the inclusion of the background interference signal in the dataset, thus further improving the quality of the analysis. High-quality MS signals were obtained from both FFPE and cytospin sample processing. A qualitative evaluation based on visual inspection and comparison of spectral data and molecular images showed better MS data quality with respect to cells prepared by cytospin with a higher ion yield  was similar to the one obtained from cells embedded with the Sakura Vip apparatus . The number of peaks per single spectrum was high in the AMO WT cells, with a median peak count of 132 in replicate 1 . Peptide profiles of AMO WT and AMO BZB shared many peaks compared to the cytospin H929 GFP/LUC, whose MS data showed a different molecular profile trend A. An ovep-values . Sixteen m/z features were overexpressed in AMO WT, from which two features  showed more than a two-fold intensity difference between the two cell lines. Ten features were instead overexpressed in AMO BZB cells, where three features  exhibited more than a four-fold intensity difference. The identification of the m/z species making these specific signatures is beyond the scope of this study because they need validation in a larger dataset. Nevertheless, these results could be reproduced on separate experiments and on a different day. Nearly identical spectra as well as equivalent peak expression were obtained in the replicate experiments, where segregation of the two different cell lines, AMO WT and AMO BZB, following AUC analysis was also obtained. Such a highly reproducible technique could be applied to other types of cells and has the potential to be a useful tool for the stratification of cell lines based on their peptidomic profile, with the goal of rapidly identify novel potential tumor molecular signatures. Comparison of the average relative intensities of the This proof-of-concept study indicates that fixation in formalin followed by centrifugation in cytospin resulted in the recovery of higher numbers of peptides compared with fixation combined with paraffin embedding. It was also shown that IMS technology holds the potential to stratify cell lines of MM, including those with a similar genetic background but different drug sensitivity. We propose this approach as an additional method for proteomic analysis of MM samples, potentially applicable to other tumor types. The continued development of proteomics and its integration with genomic studies is expected to provide valuable information for the detection of molecular mechanisms and target proteins involved in MM onset and progression.