Immunotherapy and Antivascular Targeted Therapy in Patients’ Treatment with Concurrent Malignant Tumors after Organ Transplantation: Opportunity or Challenge

Objective. To analyze the therapeutic effects and organ rejection of anti-PD-1 immunotherapy or antivascular targeting therapy on patients with combined malignancies after organ transplantation. Methods. We collected retrospective studies on “post-transplantation, cancer, immunotherapy, and vascular targeting therapy” in Embase, Wanfang database, Cochrane Library, VIP databases, CNKI, and PubMed, and the case data were organized and analyzed. Results. Data from only 40 papers met our requirements, which included 2 literature reviews, 4 original researches, and 34 case reports from 2016 to 2020. A total of 40 studies involving 66 patients were included, who were divided into 3 groups (patients using CTLA-4 inhibitors, group 1; patients who received sequential or concurrent anti-PD-1 and anti-CTLA-4 therapy, group 2; and patients using PD-1/PD-L1 inhibitors, group 3). There was no statistical difference in patients’ DCR between the three groups (P>0.05). Also, compared with group 2, there was no statistically significant difference in recipient organ rejection in group 1 and group 3 (P>0.05). The DCR rate for antivascular targeted therapy is approximately 60%. Conclusions. Immunotherapy should be carefully selected for patients with combined malignancies after organ transplantation. Antivascular targeted therapy is one of the options worth considering; the risk of side effects of drug therapy is something that needs to be closely monitored when combined with immunotherapy

Intracellular ATP concentration contributes to the cytotoxic and cytoprotective effects of adenosine.

Extracellular adenosine (ADE) interacts with cells by two pathways: by activating cell surface receptors at nanomolar/micromolar concentrations; and by interfering with the homeostasis of the intracellular nucleotide pool at millimolar concentrations. Ade shows both cytotoxic and cytoprotective effects; however, the underlying mechanisms remain unclear. In the present study, the effects of adenosine-mediated ATP on cell viability were investigated. Adenosine treatment was found to be cytoprotective in the low intracellular ATP state, but cytotoxic under the normal ATP state. Adenosine-mediated cytotoxicity and cytoprotection rely on adenosine-derived ATP formation, but not via the adenosine receptor pathway. Ade enhanced proteasome inhibition-induced cell death mediated by ATP generation. These data provide a new pathway by which adenosine exerts dual biological effects on cell viability, suggesting an important role for adenosine as an ATP precursor besides the adenosine receptor pathway

Ade decreases cell viability and induces cell death.

<p>(A) A549, MCF7, and Hela cells were exposed to Ade as indicated in normal culture medium for 72 h. Cell viability was detected using the MTS assay. Each column represents the average of five replicated experiements. Mean ±SD (n = 5). p<0.05, vs. vehicle control. (B) Ana-1 cells were treated with Ade for 72 h, cell viability was detected as in (A). Mean ±SD (n = 5). *p<0.05, **p<0.01, vs. vehicle control. (C, D, E) Ana-1 cells were incubated with Ade in normal culture medium for 12 h, then cell apoptosis was detected by either flow cytometry (FACScan; BD Biosciences) or Western blot. Representative cell death image and cell death data in Ana-1 cells are shown in (C, D). Mean ±SD (n = 3). *p<0.05, **p<0.01 vs. vehicle control. PARP cleavage is shown in (E). GAPDH was used as a loading control. (F, G) Thymus lymphocytes were incubated with Ade as indicated for 12 h, cell death was detected. Cell death images by PI staining in living cells under an inverted fluorescence microscope were shown in (F) and cell death data by flow cytometry are summarized in (G). **p<0.01 <i>vs.</i> vehicle control. Mean ±SD (n = 3).</p

Adenosine (Ade) increases intracellular ATP contents in multiple cells.

<p>(A) Primary thymocytes were exposed to either vehicle DMSO (DM) or Ade in normal culture medium for 4 h and cells were collected for total ATP assay by HPLC (LC-6AD; Shimadzu). ATP contents (µM) of equal number of cells (2×10⁵) were compared (n = 4). Each column represents the average of independent repeated experiments. Mean ±SD. *p<0.05 compared to controls. (B) K562 cells were treated with indicated doses of Ade or oligomycin (Oli; 1 µg/ml) in the absence of d-glucose in RPMI 1640 medium for 6 h. d-glucose (2 g/L) was used as a positive control. Mean ±SD (n = 4). *p<0.05 vs. control; ^#p<0.05 vs. Oli treatment alone. (C) K562 was exposed to 2 mM of Ade for 0.5, 2, and 6 h in the absence of d-glucose in the culture medium. Mean ±SD. *p<0.05 vs. 0.5 h treatment. (D) Increase of ATP in multiple cell lines: A549, MCF7, and Hela cells were exposed to either DMSO or 2 mM of Ade for 6 h in the absence (G-) or presence of d-glucose (2 g/L, G+) in the culture medium. ATP contents were detected by HPLC (n = 4) and the increase of ATP after Ade treatment was calculated as: Ade-treated/vehicle-treated. All controls were set as 1.0.</p

Oligomycin decreases proteasome inhibition-induced cell death in the presence of Ade.

<p>(A, B) K562 cells cultured in d-glucose-free medium were exposed to Ade (0.2 and 2 mM), MG132 (5 µM, or MG262 (1 µM) and their combinations; PI staining was dynamically recorded under a fluorescent microscope, typical images at 12 h are shown in (A) and (B). (C, D) K562 cells were treated with Oli (1 µM), MG132 (5 µM), or MG262 (1 µM) and their combinations in the absence or presence of Ade (2 mM) for 12 h; cell apoptosis was detected using Annexin V/PI staining. Typical images are shown in (C) and a summary of cell death is shown in (D). Mean +SD (n = 3). *p<0.05 <i>vs.</i> proteasome inhibitor treatment alone.</p

Ade increases cell viability in low ATP states and rescued cell death induced by ATP-depletion.

<p>(A, B) K562 cells were exposed to 2 mM of Ade cultured in RPMI 1640 medium with or without d-glucose for different time points. Cell density was imaged using an inverted microscope (Axio Obsever Z1; Zeiss, Germany). Typical images of cell density were selected from cells treated with Ade in d-glucose-free medium for 24 h (A) or in d-glucose-containing medium for 72 h (B). Scale bar  = 50 µm. (C) K562 cells were exposed to Ade for 24 h (left) or 48 h (right) with or without d-glucose; absolute cell numbers were counted using a cell counter. Mean ±SD (n = 3). *p<0.05 vs. d-glucose-containing cells. (D) K562 cells were incubated with Ade with or without d-glucose for 36 h. Cell viability was detected using the MTS assay. Mean ±SD (n = 3). *p<0.05 <i>vs.</i> glucose-containing cells. (E, F) K562 cells were treated with Oli with or without Ade (2 mM) in the d-glucose-free RPMI 1640 medium for 6 h, then cell apoptosis was detected by flow cytometry. Typical flow images are shown in (E) and cell death in (F). Mean ±SD (n = 3). *p<0.05 vs. Ade-treated cells. (G) K562 cells were treated with Oli (1 µg/ml) and Ade (2 mM) for 18 h in the glucose-free medium, and cells were then stained with PI and dynamically recorded under an inverted epi-fluorescent microscope. A typical image is shown. Scale bar  = 50 µM.</p

Ade transportation, but not Ade receptors, is required for Ade to exert its cytotoxic or cytoprotective effects.

<p>(A) K562 cells cultured in glucose-free culture medium were treated with Oli (0.5 µg/ml), Ade (2 mM) and DP (10 µM) for 3 h, followed by HPLC ATP assay. Mean ±SD (n = 3). *p<0.05 vs. Oli+Ade treatment. (B) K562 cells were cultured and treated in the d-glucose-free medium as indicated for 18 h, and cells were then stained with Annexin V/PI followed by flow cytometry. Viable cells are shown. Mean ±SD (n = 3). *p<0.05 each compared with Oli treatment alone; #p<0.05 each compared with Oli+Ade combination treatment. (C) K562 cells were cultured in normal medium and exposed to 2 mM Ade and DP (10 µM) for 18 h, cell numbers were counted using a cell counter. Mean ±SD (n = 3). *p<0.05 compared with vehicle control; ^#p<0.05 compared with Ade treatment alone. (D) As treated in (B), typical flow images are shown (Ade: 2 mM, Oli: 1.0 µg/ml, DP: 10 µM). (E) K562 cells were cultured in d-glucose-free medium and treated with the agents as indicated (Oli: 1.0 µg/ml, Ade: 2 mM, 8-SPT: 10 µM) for 4 h followed by ATP assay. Mean ±SD (n = 3). (F) K562 cells were cultured in normal glucose-containing medium and treated as indicated (Ade: 2 mM, 8-SPT: 10 µM) for 18 h, cell numbers were counted and summarized. Mean ±SD (n = 3). (G) K562 cells were cultured in glucose-free medium and treated as indicated (Oli: 1.0 µg/ml, 8-SPT: 10 µM) for 15 h, cell death was detected by flow cytometry. Mean ±SD (n = 3).</p

L-carnitine is an endogenous HDAC inhibitor selectively inhibiting cancer cell growth in vivo and in vitro.

L-carnitine (LC) is generally believed to transport long-chain acyl groups from fatty acids into the mitochondrial matrix for ATP generation via the citric acid cycle. Based on Warburg's theory that most cancer cells mainly depend on glycolysis for ATP generation, we hypothesize that, LC treatment would lead to disturbance of cellular metabolism and cytotoxicity in cancer cells. In this study, Human hepatoma HepG2, SMMC-7721 cell lines, primary cultured thymocytes and mice bearing HepG2 tumor were used. ATP content was detected by HPLC assay. Cell cycle, cell death and cell viability were assayed by flow cytometry and MTS respectively. Gene, mRNA expression and protein level were detected by gene microarray, Real-time PCR and Western blot respectively. HDAC activities and histone acetylation were detected both in test tube and in cultured cells. A molecular docking study was carried out with CDOCKER protocol of Discovery Studio 2.0 to predict the molecular interaction between L-carnitine and HDAC. Here we found that (1) LC treatment selectively inhibited cancer cell growth in vivo and in vitro; (2) LC treatment selectively induces the expression of p21(cip1) gene, mRNA and protein in cancer cells but not p27(kip1); (4) LC increases histone acetylation and induces accumulation of acetylated histones both in normal thymocytes and cancer cells; (5) LC directly inhibits HDAC I/II activities via binding to the active sites of HDAC and induces histone acetylation and lysine-acetylation accumulation in vitro; (6) LC treatment induces accumulation of acetylated histones in chromatin associated with the p21(cip1) gene but not p27(kip1) detected by ChIP assay. These data support that LC, besides transporting acyl group, works as an endogenous HDAC inhibitor in the cell, which would be of physiological and pathological importance

HDAC inhibitor L-carnitine and proteasome inhibitor bortezomib synergistically exert anti-tumor activity in vitro and in vivo.

Combinations of proteasome inhibitors and histone deacetylases (HDAC) inhibitors appear to be the most potent to produce synergistic cytotoxicity in preclinical trials. We have recently confirmed that L-carnitine (LC) is an endogenous HDAC inhibitor. In the current study, the anti-tumor effect of LC plus proteasome inhibitor bortezomib (velcade, Vel) was investigated both in cultured hepatoma cancer cells and in Balb/c mice bearing HepG2 tumor. Cell death and cell viability were assayed by flow cytometry and MTS, respectively. Gene, mRNA expression and protein levels were detected by gene microarray, quantitative real-time PCR and Western blot, respectively. The effect of Vel on the acetylation of histone H3 associated with the p21(cip1) gene promoter was examined by using ChIP assay and proteasome peptidase activity was detected by cell-based chymotrypsin-like (CT-like) activity assay. Here we report that (i) the combination of LC and Vel synergistically induces cytotoxicity in vitro; (ii) the combination also synergistically inhibits tumor growth in vivo; (iii) two major pathways are involved in the synergistical effects of the combinational treatment: increased p21(cip1) expression and histone acetylation in vitro and in vivo and enhanced Vel-induced proteasome inhibition by LC. The synergistic effect of LC and Vel in cancer therapy should have great potential in the future clinical trials