|Ahead of print publication
Dose-dependent cell cycle arrest and apoptosis in HER2 breast cancer cells by177Lu-CHX-A“-DTPA-Trastuzumab
Rohit Sharma1, Mythili Kameswaran2, Usha Pandey3, Ashutosh Dash3
1 Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India
2 Homi Bhabha National Institute, Mumbai, Maharashtra, India
3 Radiopharmaceuticals Division, Bhabha Atomic Research Centre; Homi Bhabha National Institute, Mumbai, Maharashtra, India
|Date of Submission||09-Jan-2019|
|Date of Decision||15-Apr-2019|
|Date of Acceptance||19-May-2019|
|Date of Web Publication||28-Jan-2020|
Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Trombay, Mumbai - 400 085, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Trastuzumab is a Food and Drug Administration-approved humanized monoclonal antibody which targets the extracellular domain of human epidermal growth factor receptor 2 (HER2) receptor overexpressed on HER2-positive breast cancer cells. The combination of Lutetium-177 (177 Lu) (t½= 6.7 days, Eβmax497 keV (78.6%) and trastuzumab makes it a suitable targeting agent for radioimmunotherapy. In preclinical and clinical studies,177 Lu-Trastuzumab has proven to be effective for the treatment of HER2-positive malignancies such as breast and ovarian cancer.
Objectives: In this study, we report the mechanism of action of177 Lu-CHX-A“-diethylenetriaminepentaacetic acid (DTPA)-trastuzumab at the cellular and molecular level by performing various in vitro assays in HER2-positive MDA-MB-453 breast cancer cells.
Materials and Methods: Trastuzumab was conjugated to the bifunctional chelating agent (BFCA) para-isothiocyanatobenzyl-DTPA and radiolabeled with177 Lu. In vitro cell binding studies were carried out in MDA-MB-453 cells to confirm the specificity of the complex toward the receptor. Cellular toxicity, cell cycle, and cell death analysis were also performed for exploring the potential of the radioimmunoconjugate at cellular and molecular level.
Results: In vitro cell binding studies showed a maximum binding of 10.7 ± 0.1% which reduced to 2.9 ± 0.1% on coincubation with unlabeled antibody. Our study revealed that the cellular toxicity was dose dependent, and mode of cell death was predominantly by apoptosis. The radioimmunoconjugate retarded the cell in the S phase of cell cycle with two-fold increase in G2/M arrest which justifies the enhanced apoptosis at higher doses.
Conclusions: The study revealed that the formulation can execute a dose-dependent cellular toxicity through induction of apoptosis.
Keywords: 177Lu-CHX-A''-DTPA-trastuzumab, apoptosis, G2/M arrest
|How to cite this URL:|
Sharma R, Kameswaran M, Pandey U, Dash A. Dose-dependent cell cycle arrest and apoptosis in HER2 breast cancer cells by177Lu-CHX-A“-DTPA-Trastuzumab. J Can Res Ther [Epub ahead of print] [cited 2020 Jul 7]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=276986
| > Introduction|| |
Among the various cancers affecting women, breast cancer is the most common one in both developed and developing countries. Approximately 20%–25% of invasive breast cancers overexpress the human epidermal growth factor receptor 2 (HER2) (HER2, also known as ErbB2/c-erbB2/HER2-neu), a transmembrane receptor tyrosine kinase,, and have the lowest prognosis among the breast cancer subtypes with low disease-free and overall survival rates, especially in disseminated cases.,,,,, The overexpression of HER2 in breast cancers has important consequences as on activation, HER2 triggers multiple downstream pathways essential for abnormal growth of cancer cells. The Food and Drug Administration-approved humanized monoclonal antibody (mAb) trastuzumab targets the extracellular domain IV of HER2 that kills the tumor cells largely by antibody-dependent cellular cytotoxicity. It has also been proposed that inhibition of the breast cancer cell proliferation is due to internalization and degradation of HER2 by promoting the activity of tyrosine kinase–ubiquitin ligase c-Cbl. Trastuzumab inhibits the mitogen-activated protein kinase and PI3K/Akt pathways which lead to an increase in cell cycle arrest and suppression of cell growth and proliferation. Although trastuzumab has significantly improved therapeutic outcomes in breast cancer patients, the benefits are limited due to patients developing primary or acquired resistance to trastuzumab.,
The overexpression of HER2 in breast cancers can be an effective target for radioimmunotherapy (RIT) wherein a mAb is coupled to a therapeutic radioisotope to form a tumor-specific targeting agent which can cause cellular toxicity. The major mode of cellular toxicity reported for ionizing radiations are apoptosis, necrosis, mitotic catastrophe, autophagy, and cell senescence depending on the nature and extent of irradiation as well as the type of cancer cells utilized.,,,,,,,, Although reports on thein vitro studies and the mechanism of action of radiolabeled antibodies are limited, understanding the mechanism of action, particularly the mode of cellular toxicity offers relevant information necessary for improving the efficacy of RIT.
Trastuzumab can be conjugated to various bifunctional chelators (BFCA) such as para-isothiocyanatobenzyl-1, 4, 7, 10-tetra-azacyclododecane-tetraacetic acid (p-NCS-Benzyl-DOTA), para-isothiocyanatobenzyl-diethylenetriaminepentaacetic acid (p-NCS-Benzyl-CHX-A“-DTPA), and their analogs, to enable labeling with177 Lu.177 Lu decays with a half-life of 6.73 days by emission of β-particles (Eβmax497 keV [78.6%]) to stable177 Hf. The emission of gamma photons with relatively low abundances (208 keV [11%]) provides the opportunity to carry out simultaneous imaging studies.
The aim here was to study the effect of treatment with different doses of177 Lu-CHX-A“-DTPA-trastuzumab on HER2-positive breast cancer cells at the cellular level and its mode of cell death on treatment. Herein, trastuzumab was conjugated to the BFCA, p–NCS-benzyl–CHX-A“-DTPA and radiolabeled with177 Lu.In vitro cell binding studies were done in MDA-MB-453 cells to confirm the specificity of the complex toward the receptor. Cellular toxicity, cell cycle, and cell death analysis were also performed for exploring the potential of the radioimmunoconjugate at cellular and molecular level. Thesein vitro studies may pave way to translation of the formulation to the clinics.
| > Materials and Methods|| |
Trastuzumab (Herceptin®) procured from Genentech, South San Francisco, was gifted by Tata Memorial Hospital, Mumbai, India, for this study.177 Lu was produced at the Dhruva reactor, BARC, Trombay, by irradiation of enriched176 Lu target followed by radiochemical processing at the Radiopharmaceuticals Division, BARC., The specific activity of177 LuCl3 produced ranged between 22 and 26 Ci/mg and is approved for clinical use by the Radiopharmaceuticals Committee which is constituted by the Department of Atomic Energy, Government of India for approving radiopharmaceutical products. p-NCS-Bn-CHX-A“-DTPA was procured from Macrocyclics (Dallas, TX, USA). Arsenazo III, yttrium chloride, and sodium bicarbonate were purchased from Sigma, USA. All other chemicals used in this study were of analytical grade and purchased from Sigma Aldrich, USA. MDA-MB-453 (HER2 positive) cell line was obtained from the National Center for Cell Sciences, Pune, India and Fetal bovine serum (FBS) from GIBCO. PD-10 columns and AMICON Ultra centrifugal filter devices (MWCO 10,000 Da) were purchased from GE Healthcare, USA and Millipore, India, respectively.
Radioactive counting was performed using a well-type NaI (Tl) gamma counter, procured from Raytest Mucha, Germany, and high-performance liquid chromatography (HPLC) analyses were performed on a Size Exclusion HPLC system (SE-HPLC) (M/s. JASCO, Japan) equipped with a TSK gel column (G3000 SWXL). The HPLC system is connected to an ultraviolet (UV) visible detector and a radioactivity detector (Raytest, Germany) for measuring absorbance and radioactivity, respectively. Phosphate buffer (0.05 M) containing 0.05% sodium azide (pH 6.8) was used as the mobile phase at 0.6 mL/min flow rate. GINA STAR software (Raytest GmBH, Germany) was used to analyze the radiochromatograms. A Chemito Spectroscan UV2600 spectrophotometer (M/s. Thermo Scientific) was used for UV absorbance measurements. Guava Flow cytometer kits were purchased from Merck KGaA (Darmstadt, Germany) for carrying out cytotoxicity assays.
Conjugation of Trastuzumab with para-isothiocyanatobenzyl diethylenetriaminepentaacetic acid
Trastuzumab was conjugated with p-NCS-CHX-A“-DTPA at 1:10 molar ratio of antibody to the BFC, as reported earlier by our group. The concentration of trastuzumab in the CHX-A“-DTPA-trastuzumab conjugate was determined by Lowry's method using IgG as the reference, and the number of CHX-A“-DTPA molecules conjugated per trastuzumab molecule was determined by spectroscopic assay using Y (III)-Arsenazo (III) complex. The basis of the spectroscopic assay is the change in absorbance occurring due to the transchelation of Y (III) from Y (III)-Arsenazo (III) complex to the CHX-A“-DTPA ligand in CHX-A“-DTPA-trastuzumab conjugate which is measured at 652 nm.
Radiolabeling of CHX-A“-DTPA-trastuzumab conjugate with Lutetium-177
Toward177 Lu labeling, CHX-A“-DTPA-trastuzumab conjugate (~ 3 mg) was taken in 0.5 mL of 0.1 M sodium acetate solution (pH 6.0). 185–370 MBq of177 LuCl3 was added to it, and the reaction mixture was incubated at ambient temperature for 15 min. The radioimmunoconjugate was purified by passing it through a PD-10 column previously equilibrated with 0.1 M sodium acetate solution (pH 6.0) and using 0.1 M sodium acetate solution (pH 6.0) as eluent. Four fractions of 2.5 mL each were collected, and the second fraction which contained the radioimmunoconjugate was used for further characterization. For determining the radiochemical purity (RCP), an aliquot of the radioimmunoconjugate was injected into the SE-HPLC system and isocratically eluted with 0.05 M phosphate buffer, pH 6.8 at a flow rate of 0.6 mL/min. Simultaneously, the RCP was also ascertained by paper chromatography (PC) on a Whatman 3 mm paper using 10 mM sodium citrate (pH 5) as the mobile phase.In vitro stability of the radioimmunoconjugate incubated at 37°C in saline was determined at various time intervals up to 5 days postpreparation by SE-HPLC as well as by PC method.
MDA-MB-453 (HER2 positive) cells were grown to 70%–80% confluence in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% FBS. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. All cellular assays were carried out in triplicates. The three sets of treatments given to the cells in all experiments comprised of cells exposed to 3.7 MBq (T1), 37 MBq (T2) of177 Lu-CHX-A“-DTPA-Trastuzumab and sets incubated with equivalent amounts of unlabeled Trastuzumab as vehicle controls (VC) (VC1 = 436 nM and VC2 = 4.36 μM), respectively.
In vitro cell-binding studies with Lutetium-177-CHX-A“-DTPA-trastuzumab
For thein vitro cell binding studies, MDA-MB-453 cells (1 × 105) were seeded in 24 well tissue culture plates and incubated overnight at 37°C. Subsequently, cells were incubated with 0.7 nM of177 Lu-CHX-A“-DTPA-trastuzumab at 37°C for 1 h. After incubation, cells were washed twice with ice cold 0.05 M phosphate buffer saline (PBS), pH 7.4, and then were solubilized with 1 mL of 1N NaOH. The solution was counted to determine the radioactivity associated with cells in a NaI (Tl) gamma counter. The percent of radioactivity bound to the cells with respect to the total radioactivity added was determined. Specific uptake was determined by coincubation of the same number of cells with177 Lu-CHX-A“-DTPA-trastuzumab and 100 fold excess of unlabeled trastuzumab. The percentage inhibition was calculated as ([difference between cell binding of radiolabeled antibody in the absence and presence of cold antibody conjugate]/cell binding in the absence of antibody conjugate) × 100.
Cellular toxicity estimation by 3- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay
For determination of cell toxicity, colorimetric 3- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay was carried out. Herein, 3 × 103 MDA-MB-453 cells were cultured per well in a 96-well plate in triplicates and incubated overnight at 37°C in a humidified 5% CO2 atmosphere. The cells treated with177 Lu-CHX-A“-DTPA-trastuzumab and unlabeled trastuzumab were incubated for 48 h at 37°C in a humidified 5% CO2 atmosphere. MDA-MB-453 cells in DMEM medium without any treatment served as the control group. At the end of incubation, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well and incubated for 3 h at 37°C. The supernatant was removed, and 150 μL of solubilizing solution (10% Triton X-100 in acidic isopropanol) was added. Absorbance in each well was measured at 570 nm with reference to 630 nm in a BioTek Universal Microplate Reader (BioTek USA, Winooski, VT). The percent cell viability was calculated as ratio of optical density of the treated samples to the control samples, multiplied by 100.
Estimation of cell viability by flow cytometer
1 × 106 MDA-MB-453 cells were plated overnight in DMEM medium containing 10% FBS. The adherent cells were grouped in triplicates and given treatments as mentioned above and incubated for a period of 48 h at 37°C in a humidified 5% CO2 atmosphere. Equivalent number of MDA-MB-453 cells without any treatment served as the control group. After the incubation period, cells were harvested by trypsinization and resuspended in 50 μL of PBS containing 1% bovine serum albumin (BSA). 250 μL of Guava via count reagent was added into each tube and incubated in dark for 15 min at room temperature (RT). Samples were acquired on a Guava EasyCyte Flow Cytometer System and analyzed with guava Soft 3.1.1.
Cell cycle analysis by flow cytometry
To synchronize the cell cycle phases, 1 × 106 MDA-MB-453 cells were starved of FBS 1 day before the study and incubated overnight at 37°C in a humidified 5% CO2 atmosphere. These cells after treatment with177 Lu-CHX-A“-DTPA-trastuzumab and equivalent amount of unlabeled trastuzumab were incubated at 37°C for 48 h in a humidified 5% CO2 atmosphere. Subsequently, the cells were washed twice with 0.05 M PBS, pH 7.4, trypsinized, harvested, and fixed in 70% ethanol for 2 h at 4°C. Cells were again washed twice with 0.05 M PBS, pH 7.4 to remove any traces of ethanol, and centrifuged. Cell pellets were resuspended in the residual PBS to which 200 μL of Guava cell cycle reagent was added, and the entire mixture was incubated for 30 min at RT in dark. Samples were then acquired on Guava EasyCyte Flow.
Study of apoptotic cell death
1 × 106 MDA-MB-453 cells treated with177 Lu-CHX-A“-DTPA-trastuzumab and unlabeled CHX-A“-DTPA-trastuzumab were harvested after trypsinization and resuspended in 100 μL of 0.05 M PBS, pH 7.4 containing 1% BSA. To this, Guava nexin reagent (100 μL) was added and incubated in dark for 20 min at RT. Samples were acquired on a Guava EasyCyte Flow Cytometer System and analyzed with Guava Soft 3.1.1.
Statistical data analysis
All data were analyzed using Origin 8.0 (Origin lab Corp. MA) statistical software. The results are expressed as mean ± standard deviation (SD) of three independent experiments. To compare the significant difference between treated and control samples, t-test was used considering P value to be 0.05 or less.
| > Results|| |
Conjugation of trastuzumab with CHX-A“-diethylenetriaminepentaacetic acid-NCS
Conjugation of trastuzumab with p-benzyl-NCS-CHX-A“-DTPA was carried out at ten times molar excess of the BFCA with respect to the antibody, as per the procedure standardized in our laboratory earlier. The number of chelator molecules per antibody molecule was determined to be four by spectroscopy assay using Y (III)-Arsenazo (III) complex. Although the presence of higher number of BFCAs per antibody molecule is advantageous as it increases radiolabeling yield, incorporation of higher number of BFCAs per antibody molecule also increases the possibility of formation of antibody-BFCA conjugate with compromised immunoreactivity, due to attachment of BFCAs at the variable region of antibody molecule. Here, the study was conducted with four BFCAs attached to one trastuzumab molecule.
Lutetium-177 labeling of CHX-A“-DTPA-trastuzumab conjugate
Near quantitative radiolabeling (>98%) of the immunoconjugate with177 Lu could be achieved on incubating the reaction mixture for 15 min at ambient temperature. The RCP of177 Lu-CHX-A“-DTPA-trastuzumab as determined by SE-HPLC was 97 ± 1%. In this HPLC system,177 Lu-CHX-A“-DTPA-trastuzumab had a retention time of 15.1 min which matched closely with the UV retention time of trastuzumab, as shown in [Figure 1]. Unbound177 Lu (III) eluted out from the SE-HPLC column at 22.0 min.
|Figure 1: SE-HPLC pattern of 177Lu-CHX-A“-DTPA-Trastuzumab. Isocratic elution performed with 0.05 M phosphate buffer containing 0.05% sodium azide, pH 6.8 with flow rate of 0.6mL/min|
Click here to view
In vitro cell-binding studies
In vitro cell-binding studies of177 Lu-CHX-A“-DTPA-trastuzumab in HER-2 expressing MDA-MB-453 breast cancer cell line showed a maximum cell uptake of 10.7 ± 0.1% while on co-incubating the cells with 177Lu-CHX-A“-DTPA-Trastuzumab and excess of unlabeled antibody (70 nM), the uptake was reduced to 2.9 ± 0.1% thereby confirming the specificity of the radioimmunoconjugate toward the HER-2 receptor [Figure 2].
|Figure 2: Cell-binding studies of Lutetium-177-CHX-A“-DTPA-trastuzumab in MDA-MB-453 cells|
Click here to view
Cellular toxicity by 3- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay
The cell viability was determined by MTT assay in which significant cell death was observed particularly at higher doses of the radioimmunoconjugate. A decrease in cell viability (76.9 ± 1.9%, [P< 0.05, n = 3]) was observed in cells treated with 37 MBq of the radioimmunoconjugate [Figure 3]-T2] as compared to cells treated with equivalent amount of cold trastuzumab (93.4 ± 1.8%) as shown in [Figure 3]-VC2. However, at a lower dose of 3.7 MBq of the radioimmunoconjugate, there was subtle inhibition of cell proliferation, and the viability was almost comparable to its corresponding VC VC1 and to the experimental control [Figure 3]-VC1].
|Figure 3: Cell toxicity of Lutetium-177-CHX-A“-DTPA-trastuzumab in MDA-MB-453 cells by [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay. T1 and T2 represent MDA-MB-453 cells treated with 3.7 and 37 MBq of Lutetium-177-CHX-A“-diethylenetriaminepentaacetic acid-trastuzumab, respectively, while vehicle controls VC1 and VC2 represent cells treated with equivalent amount of unlabeled trastuzumab present in 3.7 and 37 MBq of Lutetium-177-CHX-A“-diethylenetriaminepentaacetic acid-trastuzumab, respectively. Experimental control represents untreated MDA-MB-453 cells (P ≤ 0.05 by t-test)|
Click here to view
Cell viability assay by flow cytometry
The cell viability data obtained by MTT assay was reconfirmed by flow cytometry, wherein, the cytotoxic effect of177 Lu-CHX-A“-DTPA-trastuzumab on MDA-MB-453 cells treated with 3.7 and 37 MBq of177 Lu-CHX-A“-DTPA-trastuzumab was determined by viable cell counting using flow cytometer after 48 h of incubation. The cells treated with 37 MBq of177 Lu-CHX-A'-DTPA-trastuzumab exhibited a drastic decrease in cell viability up to 34 ± 3.5% (P< 0.05, n = 3) as compared to 82 ± 0.77% (P< 0.05, n = 3) viable cells in case of equivalent amount of cold Trastuzumab as demonstrated in [Figure 4]c and e. The cell viability in cells treated with 3.7 MBq of 177Lu-CHX-A“-DTPA-trastuzumab was 79.6 ± 0.98% (P< 0.05, n = 3) [Figure 4]d which was similar to its VC VC1 [Figure 4]b and the experimental control [Figure 4]a. [Figure 4]f represented as histogram is the percentage cell viability in response to various treatments given.
|Figure 4: Cell viability assay analyses by flow cytometry in MDA-MB-453 cells. The viability (PM2) versus forward scatter dot plot represents viable cells at the bottom of the plot and dead cells at upper region of the plot. Here, (a) represents experimental control, (b) vehicle control VC1, (c) vehicle control VC2, (d) T1-cells treated with 3.7 MBq, (e) T2-cells treated with 37 MBq of Lutetium-177-CHX-A“-DTPA-trastuzumab, (f) histogram representing percentage cell viability in response to various treatments given. This plot was made from the data obtained from (a-e) (P ≤ 0.05 by t-test)|
Click here to view
Lutetium-177-CHX-A“-DTPA-trastuzumab retards cell proliferation in S phase and induces G2 cell cycle arrest
Cell arrest was observed in our experiments. The cell cycle pattern after treatment of MD-MBA-453 cells with177 Lu-CHX-A“-DTPA-trastuzumab exhibited a positive correlation of dose with concentration of the formulation. As shown in [Table 1], the cells were retarded in S and G2/M phase of the cell cycle in dose-dependent manner. A maximum increase in G2/M arrest (9.5 ± 1.4%) was observed when cells were treated with 37 MBq177 Lu-CHX-A“-DTPA-trastuzumab as compared to its corresponding cold antibody which was 7.3 ± 0.8%. The percentage of cells in G2M phase in the experimental control was determined to be 5.8 ± 0.4% reflecting approximately two fold more G2/M arrest with the maximum concentration (4.36 μM) of177 Lu-CHX-A“-DTPA-trastuzumab. However, at lower dose of radioimmunoconjugate (3.7 MBq), the cell arrest was not significant.
|Table 1: Cell cycle analysis of 177Lu-CHX-A”-DTPA-Trastuzumab in MDA-MB-453 cell lines|
Click here to view
Mode of cell death
Data obtained through flow cytometric apoptosis analysis confirm that the mode of cell death in MD-MBA-453 after 48 h of treatment with177 Lu-CHX-A“-DTPA-Trastuzumab was predominantly by apoptosis. Apoptosis induction appears to be more pronounced at higher doses and is evident by an increase in apoptotic cell population (Annexin V+/ 7-AAD−) in a dose-dependent manner as shown in [Figure 5]a, [Figure 5]b, [Figure 5]c, [Figure 5]c, [Figure 5]d, [Figure 5]e. Maximum apoptosis was observed in cells exposed to 37 MBq of177 Lu-CHX-A“-DTPA-trastuzumab (34.6 ± 1.88%, [P< 0.05, n = 3]) when compared to its corresponding VC, VC2 (12.7 ± 0.7%, [P< 0.05, n = 3]) as demonstrated in [Figure 5]c and [Figure 5]e. Similar pattern was observed in case of necrotic or dead (Annexin V−/7-AAD+) cell populations [Figure 5]f. Unlike other assays, we observed a significant increase in apoptosis induction (23.2 ± 2.64%, [P< 0.05, n = 3]) also in case of low doses (3.7 MBq) of177 Lu-CHX-A“-DTPA-trastuzumab which was more than two fold higher than its corresponding VC VC1 [Figure 5]b, [Figure 5]c, [Figure 5]d. However, there was marginal difference in number of necrotic or dead cell population in cells treated with 3.7 MBq 177Lu-CHX-A“-DTPA-trastuzumab and its corresponding VC VC1. This is similar to the MTT and flow cytometer viability results mentioned above.
|Figure 5: Mode of cell death analyzed by flow cytometry. The 7-AAD+−/Annexin V+− dot plot represents viable cells in lower left quadrant, apoptotic cells in lower right quadrant, and dead or necrotic cells in upper right quadrant. (a) experimental control, (b) vehicle control VC1, (c) vehicle control VC2, (d) T1-cells treated with 3.7 MBq, (e) T2-cells treated with 37 MBq of Lutetium-177-CHX-A“-DTPA-trastuzumab, (f) histogram prepared from data of (a-e) (P ≤ 0.05 by t-test) indicating percentage cell populations having undergone cell death by apoptosis/necrosis in response to various treatments given|
Click here to view
| > Discussion|| |
RIT has become an effective treatment strategy for various types of cancers particularly for hematopoietic malignancies. In recent times, the availability of humanized monoclonal antibodies against various cell surface receptors overexpressed in many cancers has led to the use of RIT in others types of solid cancers as well. However, convincing therapeutic outcomes have not yet been obtained with the use of immunotherapy in patients with solid cancers. The most obvious reason being unlike lymphoma, most of the mAbs used are unable to affect tumor growth due to lower uptake and lower penetration into the tumors. Several treatment modalities are being undertaken for improving the antibody uptake and enhancing radiosensitization in cases of solid tumors, through RIT.,,
In this study, the preparation and bioevaluation of177 Lu-CHX-A“-DTPA-trastuzumab as a radioimmunotherapeutic agent targeting HER-2-positive breast solid tumors is reported. Conjugation of CHX-A“-DTPA to trastuzumab resulted in high177 Lu labeling yields in shorter time at ambient temperature. At ten times excess of the ligand to antibody, four ligand molecules were conjugated to trastuzumab sufficient to achieve high RCP of177 Lu-CHX-A“-DTPA-trastuzumab. Higher ligand to antibody ratios were not used deliberately since the conjugation of more number of ligand molecules is known to affect the immunoreactivity of the radioimmunoconjugate., A similar study was reported by Guleria et al. wherein rituximab conjugated with p-NCS-benzyl-DOTA as BFCA showed maximum immunoreactivity with lowest number of BFCAs (1.62 average number of BFCAs per antibody molecule). Thein vitro specificity of177 Lu-CHX-A“-DTPA-trastuzumab was confirmed by cell-binding studies in MDA-MB-453 breast cancer cells. Although higher cellular uptake of 2%–35% was reported with the same cell line and131 I-Trastuzumab by Kameswaran et al., the decrease in uptake with177 Lu-CHX-A“-DTPA-trastuzumab might be due to the covalent conjugation of trastuzumab with four to five molecules of BFCAs.,,
Cellular toxicity due to the β-radiations from177 Lu-CHX-A“-DTPA-trastuzumab is obvious and is contributed mainly by ionizations or energy deposition directly in, or at the cell nucleus, or very close to the DNA.,, In this work, the authors have assessed the cellular toxicity of the radioimmunoconjugate which was found to be consistent and significant. In case of MTT assay, inhibition of proliferation was observed particularly at the higher dose (37 MBq). A similar type of cell growth inhibition data by MTT assay was documented by Zhang et al. in which reduction in cell viability of 34.8% in human thyrocytes (HTori-3) was observed after treatment with131 I for 48 h. MTT assay data were reconfirmed with flow cytometry wherein similar results were obtained, and the toxicity was found to be dose dependent. Literature survey indicates thatin vitro cellular toxicity data for such radiopharmaceuticals are limited, and the results obtained here could be useful in modulating thein vivo cytotoxic radiation dose.
As mentioned above, in response to DNA damage caused by β- and γ-radiation, the molecular events within the cell which activate the G1/S and G2/M cell cycle checkpoints are essential for the maintenance of genomic stability and integrity., An evident response was observed even in the cell cycle pattern after treatment of MD-MB-453 cells with177 Lu-CHX-A“-DTPA-trastuzumab which exhibited a positive correlation with dose and concentration of the formulation [Table 1]. It has been reported that G1/S and G2/M phase retardation is induced by β- and γ-radiations. Similar type of cell cycle arrest has been reported in the human breast carcinoma cell line MCF-7 when incubated with a β-emitting radioisotope such as89 Sr which is used for bone pain palliation. Similarly, Eriksson et al. also successfully demonstrated that HeLa Hep2 cells exhibited a transient G2/M-phase arrest on incubation with a β-emitting radioisotope such as131 I. Likewise, Sharma et al. have reported two-fold increase in G2/M arrest in human osteosarcoma cell lines with188 Re-HEDP. Although the amount of G1/S and G2/M cell arrest in this study is not so pronounced even at higher doses as reported, nevertheless, these results indicate that G2/M phase arrest may be one of the possible mechanisms of the tumor growth inhibitory effect of β-emitting radioisotopes. G2/M cell cycle arrest is generally followed by apoptosis, and G2/M arrest might be the cause of cell death in MDA-MB-453 cells. However, some additional molecular evidences are needed to confirm the observation.
After determining the cytotoxicity and cell arrest, we extend our investigation in finding the mode of cell death. Although various modes of cell death such as apoptosis, necrosis, and mitotic catastrophe have been reported in literature, anticancer drugs, β-radiation, and γ irradiation have been shown to activate apoptosis pathways in leukemias and solid tumors., [Figure 5]a and [Figure 5]b show that the observed mode of cell death is predominantly by apoptosis after 48 h of treatment. Apoptosis in MDA-MB-453 cells appears to be more pronounced at higher doses of177 Lu-CHX-A“-DTPA-trastuzumab and an increase in induction of early and late apoptosis is seen in a dose-dependent manner. Zhang et al. have reported similar dose- and time-dependent increase in apoptosis induction in human thyrocytes when exposed to 7.4, 14.8, and 22.2 MBq of131 I. Likewise, Yong et al. have observed that177 Lu-Trastzumab induces apoptosis in the intraperitoneal human colon carcinoma-treated xenografts, which is comparable with ourin vitro findings. A similar study was also reported by Kumar et al. in which dose-dependent apoptosis induction and G2/M arrest was observed with177 Lu-DOTMP in human osteosarcoma (MG-63) cell lines. All these findings indicate that the major mode of cell death with therapeutic radiopharmaceuticals is by the apoptotic pathways. Understanding the mechanism of cell death not only opens the path of multimodality therapeutic avenue but also provides new targets for the development of therapeutic agents. Hence, our findings could probably be useful for combinational therapy in conjunction with other apoptosis inducing chemotherapeutic drugs. However, there may be other radiobiological phenomena that may be responsible for killing of cancer cells during radionuclide therapy, which need to be explored.
| > Conclusion|| |
Trastuzumab was radiolabeled with177 Lu through p-NCS-Bn-CHX-A''-DTPA with high RCP and was found to be specific to HER2-positive expressing cells. The cellular toxicity potential of the radiolabeled formulation was significantly higher as it was found to retard the cells in G2/M and S phase of the cell cycle and induce cell death by apoptosis in dose-dependent manner. The results of study indicate the potential of177 Lu-CHX-A“-DTPA-trastuzumab for HER-2-positive breast cancer patients.
The authors are grateful to Dr. P. K. Pujari, Associate Director, Radiochemistry and Isotope group, BARC for the support to the program. Acknowledgments are due to the staff of Radiochemicals Section, Radiopharmaceuticals Division for the regular supply of177 Lu.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Saini KS, Azim HA Jr., Metzger-Filho O, Loi S, Sotiriou C, de Azambuja E, et al.
Beyond trastuzumab: New treatment options for HER2-positive breast cancer. Breast 2011;20 Suppl 3:S20-7.
Garrett JT, Rawale S, Allen SD, Phillips G, Forni G, Morris JC, et al.
Novel engineered trastuzumab conformational epitopes demonstratein vitro
antitumor properties against HER-2/neu. J Immunol 2007;178:7120-31.
Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: Correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987;235:177-82.
Seshadri R, Firgaira FA, Horsfall DJ, McCaul K, Setlur V, Kitchen P. Clinical significance of HER-2/neu oncogene amplification in primary breast cancer. The south Australian breast cancer study group. J Clin Oncol 1993;11:1936-42.
Owens MA, Horten BC, Da Silva MM. HER2 amplification ratios by fluorescence in situ
hybridization and correlation with immunohistochemistry in a cohort of 6556 breast cancer tissues. Clin Breast Cancer 2004;5:63-9.
Tandon AK, Clark GM, Chamness GC, Ullrich A, McGuire WL. HER-2/neu oncogene protein and prognosis in breast cancer. J Clin Oncol 1989;7:1120-8.
Andrulis IL, Bull SB, Blackstein ME, Sutherland D, Mak C, Sidlofsky S, et al.
Neu/erbB-2 amplification identifies a poor-prognosis group of women with node-negative breast cancer. Toronto breast cancer study group. J Clin Oncol 1998;16:1340-9.
Paik S, Bryant J, Tan-Chiu E, Yothers G, Park C, Wickerham DL, et al.
HER2 and choice of adjuvant chemotherapy for invasive breast cancer: National surgical adjuvant breast and bowel project protocol B-15. J Natl Cancer Inst 2000;92:1991-8.
Browne BC, O'Brien N, Duffy MJ, Crown J, O'Donovan N. HER-2 signaling and inhibition in breast cancer. Curr Cancer Drug Targets 2009;9:419-38.
Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors modulatein vivo
cytotoxicity against tumor targets. Nat Med 2000;6:443-6.
Klapper LN, Waterman H, Sela M, Yarden Y. Tumor-inhibitory antibodies to HER-2/ErbB-2 may act by recruiting C-Cbl and enhancing ubiquitination of HER-2. Cancer Res 2000;60:3384-8.
Junttila TT, Akita RW, Parsons K, Fields C, Lewis Phillips GD, Friedman LS, et al.
Ligand-independent HER2/HER3/PI3K complex is disrupted by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941. Cancer Cell 2009;15:429-40.
Gajria D, Chandarlapaty S. HER2-amplified breast cancer: Mechanisms of trastuzumab resistance and novel targeted therapies. Expert Rev Anticancer Ther 2011;11:263-75.
Zhang S, Huang WC, Li P, Guo H, Poh SB, Brady SW, et al.
Combating trastuzumab resistance by targeting SRC, a common node downstream of multiple resistance pathways. Nat Med 2011;17:461-9.
Dewey WC, Ling CC, Meyn RE. Radiation-induced apoptosis: Relevance to radiotherapy. Int J Radiat Oncol Biol Phys 1995;33:781-96.
Rupnow BA, Knox SJ. The role of radiation-induced apoptosis as a determinant of tumor responses to radiation therapy. Apoptosis 1999;4:115-43.
Cragg MS, Harris C, Strasser A, Scott CL. Unleashing the power of inhibitors of oncogenic kinases through BH3 mimetics. Nat Rev Cancer 2009;9:321-6.
Sato N, Mizumoto K, Nakamura M, Ueno H, Minamishima YA, Farber JL, et al.
A possible role for centrosome overduplication in radiation-induced cell death. Oncogene 2000;19:5281-90.
Vakifahmetoglu H, Olsson M, Zhivotovsky B. Death through a tragedy: Mitotic catastrophe. Cell Death Differ 2008;15:1153-62.
Jonathan EC, Bernhard EJ, McKenna WG. How does radiation kill cells? Curr Opin Chem Biol 1999;3:77-83.
Hotchkiss RS, Strasser A, McDunn JE, Swanson PE. Cell death. N
Engl J Med 2009;361:1570-83.
Roninson IB. Tumor cell senescence in cancer treatment. Cancer Res 2003;63:2705-15.
Schmitt CA. Cellular senescence and cancer treatment. Biochim Biophys Acta 2007;1775:5-20.
Firestone R, Shirley VS, editors. Table of Isotopes. 8th
ed. New York: Wiley; 1996.
Yong KJ, Milenic DE, Baidoo KE, Brechbiel MW. Mechanisms of cell killing response from low linear energy transfer (LET) radiation originating from (177)Lu radioimmunotherapy targeting disseminated intraperitoneal tumor xenografts. Int J Mol Sci 2016;17. pii: E736.
Ray GL, Baidoo KE, Keller LM, Albert PS, Brechbiel MW, Milenic DE. Pre-clinical assessment of Lu-labeled trastuzumab targeting HER2 for treatment and management of cancer patients with disseminated intraperitoneal disease. Pharmaceuticals (Basel) 2011;5:1-5.
Knogler K, Grünberg J, Novak-Hofer I, Zimmermann K, Schubiger PA. Evaluation of 177Lu-DOTA-labeled aglycosylated monoclonal anti-L1-CAM antibody chCE7: Influence of the number of chelators on thein vitro
properties. Nucl Med Biol 2006;33:883-9.
Vimalnath KV, Shetty P, Lohar SP, Adya VC, Thulasidas SK, Chakraborty S, et al
. Aspects of yield and specific activity of (n, γ) produced177
Lu used in targeted radionuclide therapy. J Radioanal Nucl Chem 2014;302:809-12.
Dash A, Pillai MR, Knapp FF Jr. Production of (177)Lu for targeted radionuclide therapy: Available options. Nucl Med Mol Imaging 2015;49:85-107.
Kameswaran M, Pandey U, Dhakan C, Pathak K, Gota V, Vimalnath KV, et al.
Synthesis and preclinical evaluation of (177) Lu-CHX-A“-DTPA-rituximab as a radioimmunotherapeutic agent for non-Hodgkin's lymphoma. Cancer Biother Radiopharm 2015;30:240-6.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-75.
Pippin CG, Parker TA, McMurry TJ, Brechbiel MW. Spectrophotometric method for the determination of a bifunctional DTPA ligand in DTPA-monoclonal antibody conjugates. Bioconjug Chem 1992;3:342-5.
Forrer F, Chen J, Fani M, Powell P, Lohri A, Müller-Brand J, et al. In vitro
characterization of (177)Lu-radiolabelled chimeric anti-CD20 monoclonal antibody and a preliminary dosimetry study. Eur J Nucl Med Mol Imaging 2009;36:1443-52.
Meredith RF, Khazaeli MB, Plott WE, Grizzle WE, Liu T, Schlom J, et al.
Phase II study of dual 131I-labeled monoclonal antibody therapy with interferon in patients with metastatic colorectal cancer. Clin Cancer Res 1996;2:1811-8.
Meyer T, Gaya AM, Dancey G, Stratford MR, Othman S, Sharma SK, et al.
A phase I trial of radioimmunotherapy with 131I-A5B7 anti-CEA antibody in combination with combretastatin-A4-phosphate in advanced gastrointestinal carcinomas. Clin Cancer Res 2009;15:4484-92.
Kraeber-Bodéré F, Bodet-Milin C, Niaudet C, Saï-Maurel C, Moreau A, Faivre-Chauvet A, et al
. Comparative toxicity and efficacy of combined radioimmunotherapy and antiangiogenic therapy in carcinoembryonic antigen-expressing medullary thyroid cancer xenograft. J Nucl Med 2010;51:624-31.
Kukis DL, DeNardo GL, DeNardo SJ, Mirick GR, Miers LA, Greiner DP, et al.
Effect of the extent of chelate substitution on the immunoreactivity and biodistribution of 2IT-BAT-lym-1 immunoconjugates. Cancer Res 1995;55:878-84.
Guleria M, Das T, Kumar C, Sharma R, Amirdhanayagam J, Sarma HD, et al.
Effect of number of bifunctional chelating agents on the pharmacokinetics and immunoreactivity of 177Lu-labeled rituximab: A systemic study. Anticancer Agents Med Chem 2018;18:146-53.
Kameswaran M, Gota V, Ambade R, Gupta S, Dash A. Preparation and preclinical evaluation of 131 I-trastuzumab for breast cancer. J Labelled Comp Radiopharm 2017;60:12-9.
Yong KJ, Milenic DE, Baidoo KE, Brechbiel MW. (212) Pb-radioimmunotherapy induces G(2) cell-cycle arrest and delays DNA damage repair in tumor xenografts in a model for disseminated intraperitoneal disease. Mol Cancer Ther 2012;11:639-48.
Yong KJ, Milenic DE, Baidoo KE, Brechbiel MW. Sensitization of tumor to212
Pb radioimmunotherapy by gemcitabine involves initial abrogation of G2 arrest and blocked DNA damage repair by interference with Rad51. Int J Radiat Oncol Biol Phys 2013;85:1119-26.
Helleday T, Lo J, van Gent DC, Engelward BP. DNA double-strand break repair: From mechanistic understanding to cancer treatment. DNA Repair (Amst) 2007;6:923-35.
Zhang W, Gao R, Yu Y, Guo K, Hou P, Yu M, et al.
Iodine-131 induces apoptosis in HTori-3 human thyrocyte cell line and G2/M phase arrest in a p53-independent pathway. Mol Med Rep 2015;11:3148-54.
Nicolini F, Burmistrova O, Marrero MT, Torres F, Hernández C, Quintana J, et al.
Induction of G2/M phase arrest and apoptosis by the flavonoid tamarixetin on human leukemia cells. Mol Carcinog 2014;53:939-50.
Wang C, Wang J, Jiang H, Zhu M, Chen B, Bao W.In vitro
study on apoptosis induced by strontium-89 in human breast carcinoma cell line. J Biomed Biotechnol 2011;2011:541487.
Eriksson D, Blomberg J, Lindgren T, Löfroth PO, Johansson L, Riklund K, et al.
Iodine-131 induces mitotic catastrophes and activates apoptotic pathways in HeLa Hep2 cells. Cancer Biother Radiopharm 2008;23:541-9.
Sharma R, Kumar C, Mallia MB, Kameswaran M, Sarma HD, Banerjee S, et al. In vitro
evaluation of 188Re-HEDP: A mechanistic view of bone pain palliations. Cancer Biother Radiopharm 2017;32:184-91.
Topham CH, Taylor SS. Mitosis and apoptosis: How is the balance set? Curr Opin Cell Biol 2013;25:780-5.
Friesen C, Fulda S, Debatin KM. Cytotoxic drugs and the CD95 pathway. Leukemia 1999;13:1854-8.
Friesen C, Lubatschofski A, Kotzerke J, Buchmann I, Reske SN, Debatin KM. Beta-irradiation used for systemic radioimmunotherapy induces apoptosis and activates apoptosis pathways in leukaemia cells. Eur J Nucl Med Mol Imaging 2003;30:1251-61.
Kumar C, Sharma R, Das T, Korde A, Sarma H, Banerjee S, et al.
177 Lu-DOTMP induces G2/M cell cycle arrest and apoptosis in MG63 cell line. J Labelled Comp Radiopharm 2018;61:837-46.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]