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Activation of procaspase-8 in the death effector domain (DED) filaments of the death-inducing signaling complex (DISC) is a key step in apoptosis. In this study, a rationally designed cell-penetrating peptide, DEDid, was engineered to mimic the h2b helical region of procaspase-8-DED2 containing a highly conservative FL motif. Furthermore, mutations were introduced into the DEDid binding site of the procaspase-8 type I interface. Additionally, our data suggest that DEDid targets other type I DED interactions such as those of FADD. Both approaches of blocking type I DED interactions inhibited CD95L-induced DISC assembly, caspase activation and apoptosis. We showed that inhibition of procaspase-8 type I interactions by mutations not only diminished procaspase-8 recruitment to the DISC but also destabilized the FADD core of DED filaments. Taken together, this study offers insights to develop strategies to target DED proteins, which may be considered in diseases associated with cell death and inflammation.
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Procaspase-8 is a key mediator of death receptor (DR)-mediated pathways. Recently, the role of post-translational modifications (PTMs) of procaspase-8 in controlling cell death has received increasing attention. Here, using mass spectrometry screening, pharmacological inhibition and biochemical assays, we show that procaspase-8 can be targeted by the PRMT5/RIOK1/WD45 methylosome complex. Furthermore, two potential methylation sites of PRMT5 on procaspase-8, R233 and R435, were identified in silico. R233 and R435 are highly conserved in mammals and their point mutations are among the most common mutations of caspase-8 in cancer. The introduction of mutations at these positions resulted in inhibitory effects on CD95L-induced caspase-8 activity, effector caspase activation and apoptosis. In addition, we show that procaspase-8 can undergo symmetric di-methylation. Finally, the pharmacological inhibition of PRMT5 resulted in the inhibitory effects on caspase activity and apoptotic cell death. Taken together, we have unraveled the additional control checkpoint in procaspase-8 activation and the arginine methylation network in the extrinsic apoptosis pathway.
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Apoptosis , Arginina , Caspasa 8 , Proteína-Arginina N-Metiltransferasas , Caspasa 8/metabolismo , Caspasa 8/genética , Arginina/metabolismo , Humanos , Metilación , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Procesamiento Proteico-PostraduccionalRESUMEN
The importance of post-translational modifications (PTMs), particularly O-GlcNAcylation, of cytoplasmic proteins in apoptosis has been neglected for quite a while. Modification of cytoplasmic proteins by a single N-acetylglucosamine sugar is a dynamic and reversible PTM exhibiting properties more like phosphorylation than classical O- and N-linked glycosylation. Due to the sparse information existing, we have only limited understanding of how GlcNAcylation affects cell death. Deciphering the role of GlcNAcylation in cell fate may provide further understanding of cell fate decisions. This review focus on the modulation of extrinsic apoptotic pathway via GlcNAcylation carried out by O-GlcNAc transferase (OGT) or by other bacterial effector proteins.
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RL2 (recombinant lactaptin 2), a recombinant analogon of the human milk protein Κ-Casein, induces mitophagy and cell death in breast carcinoma cells. Furthermore, RL2 was shown to enhance extrinsic apoptosis upon long-term treatment while inhibiting it upon short-term stimulation. However, the effects of RL2 on the action of chemotherapeutic drugs that induce the intrinsic apoptotic pathway have not been investigated to date. Here, we examined the effects of RL2 on the doxorubicin (DXR)-induced cell death in breast cancer cells with three different backgrounds. In particular, we used BT549 and MDA-MB-231 triple-negative breast cancer (TNBC) cells, T47D estrogen receptor alpha (ERα) positive cells, and SKBR3 human epidermal growth factor receptor 2 (HER2) positive cells. BT549, MDA-MB-231, and T47D cells showed a severe loss of cell viability upon RL2 treatment, accompanied by the induction of mitophagy. Furthermore, BT549, MDA-MB-231, and T47D cells could be sensitized towards DXR treatment with RL2, as evidenced by loss of cell viability. In contrast, SKBR3 cells showed almost no RL2-induced loss of cell viability when treated with RL2 alone, and RL2 did not sensitize SKBR3 cells towards DXR-mediated loss of cell viability. Bioinformatic analysis of gene expression showed an enrichment of genes controlling metabolism in SKBR3 cells compared to the other cell lines. This suggests that the metabolic status of the cells is important for their sensitivity to RL2. Taken together, we have shown that RL2 can enhance the intrinsic apoptotic pathway in TNBC and ERα-positive breast cancer cells, paving the way for the development of novel therapeutic strategies.
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Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Receptor alfa de Estrógeno , Línea Celular Tumoral , Apoptosis , Doxorrubicina/farmacologíaRESUMEN
Hepatocellular carcinoma (HCC) has been associated with hepatitis C viral (HCV) infection as a potential risk factor. Nonetheless, the precise genetic regulatory mechanisms triggered by the virus, leading to virus-induced hepatocarcinogenesis, remain unclear. We hypothesized that HCV proteins might modulate the activity of aberrantly methylated HCC genes through regulatory pathways. Virus-host regulatory pathways, interactions between proteins, gene expression, transport, and stability regulation, were reconstructed using the ANDSystem. Gene expression regulation was statistically significant. Gene network analysis identified four out of 70 HCC marker genes whose expression regulation by viral proteins may be associated with HCC: DNA-binding protein inhibitor ID - 1 (ID1), flap endonuclease 1 (FEN1), cyclin-dependent kinase inhibitor 2A (CDKN2A), and telomerase reverse transcriptase (TERT). It suggested the following viral protein effects in HCV/human protein heterocomplexes: HCV NS3(p70) protein activates human STAT3 and NOTC1; NS2-3(p23), NS5B(p68), NS1(E2), and core(p21) activate SETD2; NS5A inhibits SMYD3; and NS3 inhibits CCN2. Interestingly, NS3 and E1(gp32) activate c-Jun when it positively regulates CDKN2A and inhibit it when it represses TERT. The discovered regulatory mechanisms might be key areas of focus for creating medications and preventative therapies to decrease the likelihood of HCC development during HCV infection.
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Carcinoma Hepatocelular , Hepatitis C , Neoplasias Hepáticas , Virosis , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatitis C/complicaciones , Hepatitis C/genética , N-Metiltransferasa de Histona-LisinaRESUMEN
Breast cancer is still the most common cancer in women worldwide. Resistance to drugs and recurrence of the disease are two leading causes of failure in treatment. For a more efficient treatment of patients, the development of novel therapeutic regimes is needed. Recent studies indicate that modulation of autophagy in concert with apoptosis induction may provide a promising novel strategy in breast cancer treatment. Apoptosis and autophagy are two tightly regulated distinct cellular processes. To maintain tissue homeostasis abnormal cells are disposed largely by means of apoptosis. Autophagy, however, contributes to tissue homeostasis and cell fitness by scavenging of damaged organelles, lipids, proteins, and DNA. Defects in autophagy promote tumorigenesis, whereas upon tumor formation rapidly proliferating cancer cells may rely on autophagy to survive. Given that evasion of apoptosis is one of the characteristic hallmarks of cancer cells, inhibiting autophagy and promoting apoptosis can negatively influence cancer cell survival and increase cell death. Hence, combination of antiautophagic agents with the enhancement of apoptosis may restore apoptosis and provide a therapeutic advantage against breast cancer. In this review, we discuss the cross-talk of autophagy and apoptosis and the diverse facets of autophagy in breast cancer cells leading to novel models for more effective therapeutic strategies.
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Autofagia , Neoplasias de la Mama , Apoptosis , Neoplasias de la Mama/genética , Línea Celular Tumoral , Supervivencia Celular , Femenino , HumanosRESUMEN
The extrinsic pathway is mediated by death receptors (DRs), including CD95 (APO-1/Fas) or TRAILR-1/2. Defects in apoptosis regulation lead to cancer and other malignancies. The master regulator of the DR networks is the cellular FLICE inhibitory protein (c-FLIP). In addition to its key role in apoptosis, c-FLIP may exert other cellular functions, including control of necroptosis, pyroptosis, nuclear factor κB (NF-κB) activation, and tumorigenesis. To gain further insight into the molecular mechanisms of c-FLIP action in cancer networks, we focus on the structure, isoforms, interactions, and post-translational modifications of c-FLIP. We also discuss various avenues to target c-FLIP in cancer cells for therapeutic benefit.
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Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Neoplasias , Apoptosis/fisiología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Transducción de Señal , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
CD95/Fas/APO-1 can trigger apoptotic as well as nonapoptotic pathways in immune cells. CD95 signaling in humans can be inhibited by several mechanisms, including mutations in the gene encoding CD95. CD95 mutations lead to autoimmune disorders, such as autoimmune lymphoproliferative syndrome (ALPS). Gaining further insight into the reported mutations of CD95 and resulting alterations of its signaling networks may provide further understanding of their presumed role in certain autoimmune diseases. For illustrative purposes and to better understand the potential outcomes of CD95 mutations, here we assign their positions to the recently determined 3D structures of human CD95. Based on this, we make certain predictions and speculate on the putative role of CD95 mutation defects in CD95-mediated signaling for certain autoimmune diseases.
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Enfermedades Autoinmunes , Trastornos Linfoproliferativos , Receptor fas/genética , Apoptosis/genética , Enfermedades Autoinmunes/genética , Autoinmunidad/genética , Muerte Celular/genética , Humanos , Trastornos Linfoproliferativos/genética , Mutación/genética , Receptor fas/metabolismoRESUMEN
Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (TRAIL-R1/R2). Stimulation of these receptors with their cognate ligands leads to the assembly of the death-inducing signaling complex (DISC). DISC comprises DR, the adaptor protein Fas-associated protein with death domain (FADD), procaspases-8/-10, and cellular FADD-like interleukin (IL)-1ß-converting enzyme-inhibitory proteins (c-FLIPs). The DISC serves as a platform for procaspase-8 processing and activation. The latter occurs via its dimerization/oligomerization in the death effector domain (DED) filaments assembled at the DISC. Activation of procaspase-8 is followed by its processing, which occurs in several steps. In this work, an established experimental workflow is described that allows the measurement of DISC formation and the processing of procaspase-8 in this complex. The workflow is based on immunoprecipitation techniques supported by western blot analysis. This workflow allows careful monitoring of different steps of procaspase-8 recruitment to the DISC and its processing and is highly relevant for investigating molecular mechanisms of extrinsic apoptosis.
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Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte , Receptor fas , Apoptosis , Caspasa 8/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Transducción de Señal , Receptor fas/metabolismoRESUMEN
The development of efficient combinatorial treatments is one of the key tasks in modern anti-cancer therapies. An apoptotic signal can either be induced by activation of death receptors (DR) (extrinsic pathway) or via the mitochondria (intrinsic pathway). Cancer cells are characterized by deregulation of both pathways. Procaspase-8 activation in extrinsic apoptosis is controlled by c-FLIP proteins. We have recently reported the small molecules FLIPinB/FLIPinBγ targeting c-FLIPL in the caspase-8/c-FLIPL heterodimer. These small molecules enhanced caspase-8 activity in the death-inducing signaling complex (DISC), CD95L/TRAIL-induced caspase-3/7 activation and subsequent apoptosis. In this study to increase the pro-apoptotic effects of FLIPinB/FLIPinBγ and enhance its therapeutic potential we investigated costimulatory effects of FLIPinB/FLIPinBγ in combination with the pharmacological inhibitors of the anti-apoptotic Bcl-2 family members such as ABT-263 and S63845. The combination of these inhibitors together with FLIPinB/FLIPinBγ increased CD95L-induced cell viability loss, caspase activation and apoptosis. Taken together, our study suggests new approaches for the development of combinatorial anti-cancer therapies specifically targeting both intrinsic and extrinsic apoptosis pathways.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Compuestos de Anilina/farmacología , Caspasa 8/metabolismo , Supervivencia Celular/efectos de los fármacos , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Sistemas de Liberación de Medicamentos , Proteína Ligando Fas/farmacología , Células HeLa , Humanos , Sulfonamidas/farmacologíaRESUMEN
The current pandemic of novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) poses a significant global public health threat. While urgent regulatory measures in control of the rapid spread of this virus are essential, scientists around the world have quickly engaged in this battle by studying the molecular mechanisms and searching for effective therapeutic strategies against this deadly disease. At present, the exact mechanisms of programmed cell death upon SARS-CoV-2 infection remain to be elucidated, though there is increasing evidence suggesting that cell death pathways play a key role in SARS-CoV-2 infection. There are several types of programmed cell death, including apoptosis, pyroptosis, and necroptosis. These distinct programs are largely controlled by the proteins of the death domain (DD) superfamily, which play an important role in viral pathogenesis and host antiviral response. Many viruses have acquired the capability to subvert the program of cell death and evade the host immune response, mainly by virally encoded gene products that control cell signaling networks. In this mini-review, we will focus on SARS-CoV-2, and discuss the implication of restraining the DD-mediated signaling network to potentially suppress viral replication and reduce tissue damage.
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Cell fate decisions regulating survival and death are essential for maintaining tissue homeostasis; dysregulation thereof can lead to tumor development. In some cases, survival and death are triggered by the same receptor, e.g., tumor necrosis factor (TNF)-receptor 1 (TNFR1). We identified a prominent role for the cold shock Y-box binding protein-1 (YB-1) in the TNF-induced activation and nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65. In the absence of YB-1, the expression of TNF receptor-associated factor 2 (TRAF2), a central component of the TNF receptor signaling complex required for NF-κB activation, is significantly reduced. Therefore, we hypothesized that the loss of YB-1 results in a destabilization of TRAF2. Consistent with this hypothesis, we observed that YB-1-deficient cells were more prone to TNF-induced apoptotic cell death. We observed enhanced effector caspase-3 activation and could successfully rescue the cells using the pan-caspase inhibitor zVAD-fmk, but not necrostatin-1. Taken together, our results indicate that YB-1 plays a central role in promoting cell survival through NF-κB activation and identifies a novel mechanism by which enhanced YB-1 expression may contribute to tumor development.
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Breast cancer is still one of the most common cancers for women. Specified therapeutics are indispensable for optimal treatment. In previous studies, it has been shown that RL2, the recombinant fragment of human κ-Casein, induces cell death in breast cancer cells. However, the molecular mechanisms of RL2-induced cell death remain largely unknown. In this study, mechanisms of RL2-induced cell death in breast cancer cells were systematically investigated. In particular, we demonstrate that RL2 induces loss of mitochondrial membrane potential and cellular ATP loss followed by cell death in breast cancer cells. The mass spectrometry-based screen for RL2 interaction partners identified mitochondrial import protein TOM70 as a target of RL2, which was subsequently validated. Further to this, we show that RL2 is targeted to mitochondria after internalization into the cells, where it can also be found in the dimeric form. The importance of TOM70 and RL2 interaction in RL2-induced reduction in ATP levels was validated by siRNA-induced downregulation of TOM70, resulting in the partial rescue of ATP production. Taken together, this study demonstrates that RL2-TOM70 interaction plays a key role in RL2-mediated cell death and targeting this pathway may provide new therapeutic options for treating breast cancer.
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Apoptosis is a form of programmed cell death, deregulation of which occurs in multiple disorders, including neurodegenerative and autoimmune diseases as well as cancer. The formation of a death-inducing signaling complex (DISC) and death effector domain (DED) filaments are critical for initiation of the extrinsic apoptotic pathway. Post-translational modifications (PTMs) of DED-containing DISC components such as FADD, procaspase-8, and c-FLIP comprise an additional level of apoptosis regulation, which is necessary to overcome the threshold for apoptosis induction. In this review we discuss the influence of PTMs of FADD, procaspase-8, and c-FLIP on DED filament assembly and cell death induction, with a focus on the 3D organization of the DED filament.
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Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Caspasa 8/metabolismo , Muerte Celular , Humanos , Modelos Biológicos , Transducción de SeñalRESUMEN
Pharmacological targeting via small molecule-based chemical probes has recently acquired an emerging importance as a valuable tool to delineate molecular mechanisms. Induction of apoptosis via CD95/Fas and TRAIL-R1/2 is triggered by the formation of the death-inducing signaling complex (DISC). Caspase-8 activation at the DISC is largely controlled by c-FLIP proteins. However molecular mechanisms of this control have just started to be uncovered. In this study we report the first-in-class chemical probe targeting c-FLIPL in the heterodimer caspase-8/c-FLIPL. This rationally designed small molecule was aimed to imitate the closed conformation of the caspase-8 L2' loop and thereby increase caspase-8 activity after initial processing of the heterodimer. In accordance with in silico predictions, this small molecule enhanced caspase-8 activity at the DISC, CD95L/TRAIL-induced caspase activation, and subsequent apoptosis. The generated computational model provided further evidence for the proposed effects of the small molecule on the heterodimer caspase-8/c-FLIPL. In particular, the model has demonstrated that boosting caspase-8 activity by the small molecule at the early time points after DISC assembly is crucial for promoting apoptosis induction. Taken together, our study allowed to target the heterodimer caspase-8/c-FLIPL and get new insights into molecular mechanisms of its activation.
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Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasa 8/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Multimerización de Proteína , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/química , Caspasa 8/química , Línea Celular Tumoral , Supervivencia Celular , Evaluación Preclínica de Medicamentos , Proteína Ligando Fas , Humanos , Modelos Moleculares , Reproducibilidad de los Resultados , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismoRESUMEN
A recombinant fragment of human κ-Casein, termed RL2, induces cell death of breast cancer cells; however, molecular mechanisms of RL2-mediated cell death have remained largely unknown. In the current study, we have decoded the molecular mechanism of the RL2-mediated cell death and found that RL2 acts via the induction of mitophagy. This was monitored by the loss of adenosine triphosphate production, LC3B-II generation, and upregulation of BNIP3 and BNIP3L/NIX, as well as phosphatase and tensin homolog-induced kinase 1. Moreover, we have analyzed the cross talk of this pathway with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis upon combinatorial treatment with RL2 and TRAIL. Strikingly, we found two opposite effects of this co-treatment. RL2 had inhibitory effects on TRAIL-induced cell death upon short-term co-stimulation. In particular, RL2 treatment blocked TRAIL-mediated caspase activation, cell viability loss, and apoptosis, which was mediated via the downregulation of the core proapoptotic regulators. Contrary to short-term co-treatment, upon long-term co-stimulation, RL2 sensitized the cells toward TRAIL-induced cell death; the latter observation provides the basis for the development of therapeutic approaches in breast cancer cells. Collectively, our findings have important implications for cancer therapy and reveal the molecular switches of the cross talk between RL2-induced mitophagy and TRAIL-mediated apoptosis.
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The assembly of the death-inducing signaling complex (DISC) and death effector domain (DED) filaments at CD95/Fas initiates extrinsic apoptosis. Procaspase-8 activation at the DED filaments is controlled by short and long c-FLIP isoforms. Despite apparent progress in understanding the assembly of CD95-activated platforms and DED filaments, the detailed molecular mechanism of c-FLIP action remains elusive. Here, we further addressed the mechanisms of c-FLIP action at the DISC using biochemical assays, quantitative mass spectrometry, and structural modeling. Our data strongly indicate that c-FLIP can bind to both FADD and procaspase-8 at the DED filament. Moreover, the constructed in silico model shows that c-FLIP proteins can lead to the formation of the DISCs comprising short DED filaments as well as serve as bridging motifs for building a cooperative DISC network, in which adjacent CD95 DISCs are connected by DED filaments. This network is based on selective interactions of FADD with both c-FLIP and procaspase-8. Hence, c-FLIP proteins at the DISC control initiation, elongation, and composition of DED filaments, playing the role of control checkpoints. These findings provide new insights into DISC and DED filament regulation and open innovative possibilities for targeting the extrinsic apoptosis pathway.
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Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Dominio Efector de Muerte , Secuencia de Aminoácidos , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/química , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Células HeLa , Humanos , Células Jurkat , Modelos Moleculares , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Receptor fas/metabolismoRESUMEN
BACKGROUND: Structural homology modeling supported by bioinformatics analysis plays a key role in uncovering new molecular interactions within gene regulatory networks. Here, we have applied this powerful approach to analyze the molecular interactions orchestrating death receptor signaling networks. In particular, we focused on the molecular mechanisms of CD95-mediated NF-κB activation and the role of c-FLIP/NEMO interaction in the induction of this pathway. RESULTS: To this end, we have created the homology model of the c-FLIP/NEMO complex using the reported structure of the v-FLIP/NEMO complex, and rationally designed peptides targeting this complex. The designed peptides were based on the NEMO structure. Strikingly, the experimental in vitro validation demonstrated that the best inhibitory effects on CD95-mediated NF-κB activation are exhibited by the NEMO-derived peptides with the substitution D242Y of NEMO. Furthermore, we have assumed that the c-FLIP/NEMO complex is recruited to the DED filaments formed upon CD95 activation and validated this assumption in silico. Further insight into the function of c-FLIP/NEMO complex was provided by the analysis of evolutionary conservation of interacting regions which demonstrated that this interaction is common in distinct mammalian species. CONCLUSIONS: Taken together, using a combination of bioinformatics and experimental approaches we obtained new insights into CD95-mediated NF-κB activation, providing manifold possibilities for targeting the death receptor network.
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Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Quinasa I-kappa B/metabolismo , Sondas Moleculares , FN-kappa B/metabolismo , Receptor fas/metabolismo , Secuencia de Aminoácidos , Biología Computacional , Humanos , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Alineación de Secuencia , Transducción de SeñalRESUMEN
Autophagy is a degradative process in which cellular organelles and proteins are recycled to restore homeostasis and cellular metabolism. Autophagy can be either a prosurvival or a prodeath process and remains one of the most fundamental processes for cell vitality. Thus autophagy modulation is an important approach for reinforcement anticancer therapeutics. Earlier we have demonstrated that recombinant analog of human milk protein lactaptin (RL2) induced apoptosis of various cultured cancer cells and activated lipidation of microtubule-associated protein 1 light chain 3 (LC3). In this study we investigated whether autophagy inhibitors-chloroquine (CQ), Ku55933 (Ku), and 3-methyladenine (3MA)-or inducer-rapamycin (Rap)-can enhance cytotoxic activity of lactaptin analog in cancer cells and its anticancer activity in the mice model. Western Blot analysis revealed that RL2 induced short-term autophagy in MDA-MB-231 and MCF-7 cells at early stages of incubation and that these data were confirmed by the transmission electron microscopy of autophagosome/autophagolysosome formation. RL2 stimulates reactive oxygen species (ROS) production, autophagosomes accumulation, upregulation of ATG5 with processing of LC3I to LC3II, and downregulation of p62/sequestosome 1 (p62). We have shown that autophagy modulators, CQ, Ku, and Rap, synergistically increased cytotoxicity of RL2, and RL2 with CQ induced autophagic cell death. In addition, CQ, Ku, and Rap in combination with RL2 decreased activity of lysosomal protease Cathepsin D. More importantly, combining RL2 with CQ, we improved antitumor effect in mice. Detected synergistic cytotoxic effects of both types of autophagy regulators, inhibitors, and inducers with RL2 against cancer cells allow us to believe that these combinations can be a basis for the new anticancer approach. Finally, we suppose that CQ and Rap promoting of short-term RL2-induced autophagy interlinks with final autophagic cell death.