Anti-tumor activity of N-thiolated β-lactam antibiotics
Introduction
Apoptosis, or programmed cell death, is an evolutionarily conserved cellular suicide program essential for the development of multi-cellular organisms [1]. By removing unwanted cells, apoptosis plays a vital role in development, tissue homeostasis, and defense against viral infections and mutations. In many human diseases, the genes controlling the apoptotic process are suppressed, over-expressed or altered by mutations [2]. Importantly, the vast majority of human cancers are associated with mutations in various apoptotic checkpoint proteins, or tumor suppressor genes. A preponderance of epidemiological and molecular evidence suggests that these mutations play a critical role in enabling the progression of malignancy through suppression of the apoptotic process [3].
The three fundamental steps of apoptosis are initiation, commitment, and execution [4]. Several apoptotic stimuli, such as irreparable DNA damage, have been shown to activate the cellular caspase enzymatic cascade pathway, which is a central part of apoptosis. It is believed that the subsequent proteolytic cleavage of a variety of intracellular substrates, such as poly(ADP-ribose) polymerase (PARP) and the retinoblastoma (Rb) protein, by effector caspases leads to the terminal hallmarks of apoptosis, such as nuclear blebbing [5], [6], [7], [8].
Selective activation of apoptotic pathways in cancer cells, but not in normal ones, is currently being pursued as a novel strategy for cancer treatment. It has been shown that standard cytotoxic chemotherapeutic drugs induce apoptosis not only in malignant but also in normal cells, while more aggressive cancer cells become resistant. This narrow therapeutic window often necessitates aggressive dosing regimens which are frequently discontinued due to toxic side effects. Therefore, it is essential to develop targeted therapies that minimize toxic side effects by specifically targeting and destroying tumors without harming non-malignant tissue.
Small synthetic molecules with apoptosis-inducing ability have potential to be developed into novel chemotherapeutic drugs. These molecules can be easily synthesized and structurally manipulated for selective development [9]. For more than 60 years, β-lactam antibiotics have played an essential role in treating bacterial infections [10], [11]. These β-lactam drugs selectively disrupt the formation of bacterial cell walls, while eukaryotic cells are not affected, making β-lactam therapy safe for the patients. Recently, a new class of N-thiolated β-lactams was found to inhibit growth in methicillin-resistant Staphylococcus aureus[12], [13], [14], [15]. Subsequent work has demonstrated that these N-thiolated β-lactams are capable of inducing apoptosis in tumor cells, making them good candidates for anti-cancer drugs [9].
We have previously reported that β-lactam L-1 (Fig. 1A), the most potent of the N-thiolated β-lactams tested, induces DNA damage, inhibits DNA replication and activates the apoptosis in cultured human tumor cells in a time- and concentration-dependent manner [9]. We have also demonstrated that L-1 selectively induces apoptosis in leukemic Jurkat T over normal immortalized YT cells and that L-1 induces apoptosis in several solid human tumor cell lines [9]. However, the in vivo effect of these β-lactams has not been investigated yet.
In the current study, we report two major in vivo findings. First, by using subcutaneous xenografts of human breast cancer MDA-MB-231 cells implanted in nude mice, we show that L-1 inhibits tumor growth in vivo in a dose-dependent manner. In addition, we demonstrate that the anti-tumor activity of L-1 is associated with its abilities to induce DNA damage and apoptosis in tumor tissues, suggesting a potential molecular mechanism.
Section snippets
Materials
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma–Aldrich (St. Louis, MO, USA). The APO-DIRECT kit for terminal deoxynucleotidyl transferase-mediated UTP nick-end labeling (TUNEL) was purchased from BD Biosciences (San Jose, CA, USA). Antibodies against actin, heat shock protein 70 (Hsp70), DNA damage inducible gene-45β (GADD45β), anti-goat and anti-mouse IgG-horseradish peroxidase were obtained from Santa Cruz
Treatment with L-1 increases levels of GADD45β and Hsp70 proteins in leukemia Jurkat T cells
We have previously reported that N-thiolated β-lactams have the ability to induce DNA strand breaks selectively in transformed and malignant, but not in non-transformed cells [18]. We hypothesized that β-lactam-induced DNA damage should induce expression of many genes that are involved in DNA repair. To test this hypothesis, we treated human leukemia Jurkat T cells with 60 μmol/L of L-1 for various hours, followed by measuring protein levels of GADD45β and Hsp70, both of which play important
Discussion
Since many currently used anti-cancer therapies are toxic and eventually cause resistance to the treatment, researchers are attempting to search for new anti-cancer agents with little or no toxicity. It is generally believed that an important property of a potential anti-cancer drug is its ability to induce apoptosis, preferably in a highly selective manner in cancer over normal cells. It has been shown that antibiotic therapies typically use the unique molecular targets of microbes, which
Conflicts of interest
All authors declare that we have no conflicts of interest.
Acknowledgments
The authors thank Department of Defense Breast Cancer Research Program Awards (W81XWH-04-1-0688 and DAMD17-03-1-0175 to Q.P.D.) for support of this research. We also acknowledge the Karmanos Cancer Institute Pathology Core Facility for assisting in TUNEL and immunohistochemistry assays.
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2020, European Journal of Medicinal ChemistryCitation Excerpt :It is generally believed that proteolytic cleavage of a variety of intracellular substrates, including poly(ADP-ribose) polymerase and the retinoblastoma protein, by effector caspases leads to apoptosis [173]. Dou et al. reported a series of N-thiolated β-lactam derivatives as apoptosis inducer [174–177]. β-lactam 69 with an N-methylthio group was able to induce DNA damage and inhibit DNA replication in cancer cells (Fig. 15).
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